EP4210725A1 - Anticorps contenant des acides aminés non naturels et procédés de fabrication et d'utilisation de ceux-ci - Google Patents

Anticorps contenant des acides aminés non naturels et procédés de fabrication et d'utilisation de ceux-ci

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Publication number
EP4210725A1
EP4210725A1 EP21867714.4A EP21867714A EP4210725A1 EP 4210725 A1 EP4210725 A1 EP 4210725A1 EP 21867714 A EP21867714 A EP 21867714A EP 4210725 A1 EP4210725 A1 EP 4210725A1
Authority
EP
European Patent Office
Prior art keywords
amino acid
seq
uaa
position corresponding
acid sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21867714.4A
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German (de)
English (en)
Inventor
James Sebastian ITALIA
Nikolaos BIRIS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Brickbio Inc
Original Assignee
Brickbio Inc
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Filing date
Publication date
Application filed by Brickbio Inc filed Critical Brickbio Inc
Publication of EP4210725A1 publication Critical patent/EP4210725A1/fr
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6853—Carcino-embryonic antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • proteins are produced in cells via processes known as transcription and translation.
  • transcription a gene comprising a series of codons that collectively encode a protein of interest is transcribed into messenger RNA (mRNA).
  • mRNA messenger RNA
  • a ribosome attaches to and moves along the mRNA and incorporates specific amino acids into a polypeptide chain being synthesized (translated) from the mRNA at positions corresponding to the codons to produce the protein.
  • tRNAs transfer RNAs
  • tRNAs which contain an anti-codon sequence, hybridize to their respective codon sequences in mRNA and transfer the amino acid they are carrying into the nascent protein chain at the appropriate position as the protein is synthesized.
  • eukaryotic cells e.g., mammalian cells
  • the proteins may be more readily produced in a properly folded and fully active form and/or post-translationally modified in a manner similar to the native protein naturally produced in a mammalian cell.
  • UAAs unnatural amino acids
  • the core elements required for this technology include: an engineered tRNA, an engineered aminoacyl-tRNA synthetase (aaRS) that charges the tRNA with a UAA, and a unique codon, e.g., a stop codon, directing the incorporation of the UAA into the protein as it is being synthesized.
  • aaRS engineered aminoacyl-tRNA synthetase
  • a unique codon e.g., a stop codon
  • an engineered tRNA/aaRS pair derived from an organism in different domain of life as the expression host cell so as to maximize the orthogonality between the engineered tRNA/aaRS pair (e.g., an engineered bacterial tRNA/aaRS pair) and the tRNA/aaRS pairs naturally found in the expression host cell (e.g., mammalian cell).
  • the engineered tRNA which is charged with the UAA via the aaRS, binds or hybridizes to the unique codon, such as a premature stop codon (UAG, UGA, UAA) present in the mRNA encoding the protein to be expressed.
  • FIGURE 1A shows the synthesis of a protein using an endogenous tRNA and an endogenous aaRS from the expression host cell and an engineered orthogonal tRNA and an orthogonal aaRS introduced into the host cell so as to facilitate the incorporation of a UAA into the protein as it is synthesized via the ribosome.
  • orthogonal tRNA/aaRS pairs have been produced for certain of the naturally occurring amino acids (see, e.g., U.S. Patent Publication US2017/0349891, and Zheng et al. (2016) BIOCHEM.57:441-445).
  • the approach facilitates the expression of proteins containing site specific modifications such as bioconjugation handles and photoactivatable crosslinkers, which can be used as therapeutics (e.g., antibody drug conjugates (ADCs), bi-specific antibodies (e.g., bispecific monoclonal antibodies), nanobodies, chemokines, vaccines, coagulation factors, hormones, and enzymes).
  • ADCs antibody drug conjugates
  • bi-specific antibodies e.g., bispecific monoclonal antibodies
  • nanobodies e.g., chemokines, vaccines, coagulation factors, hormones, and enzymes.
  • the invention is based, in part, on the discovery of specific locations in antibodies that allow for efficient incorporation of one, or more, unnatural amino acids (UAAs) into the antibodies.
  • UAAs unnatural amino acids
  • the invention is further based, in part, on the discovery of combinations of specified positions in antibodies, UAAs for incorporation at those positions, and molecules for conjugation at those UAAs.
  • the invention provides an antibody comprising (e.g., in a heavy chain or a fragment thereof): (i) the amino acid sequence of SEQ ID NO: 118, with an unnatural amino acid (UAA) at a position corresponding to P6 of SEQ ID NO: 118; the amino acid sequence of SEQ ID NO: 119, with an unnatural amino acid (UAA) at a position corresponding to P14 of SEQ ID NO: 119; the amino acid sequence of SEQ ID NO: 120, with an unnatural amino acid (UAA) at a position corresponding to P14 of SEQ ID NO: 120; or the amino acid sequence of SEQ ID NO: 121, with an unnatural amino acid (UAA) at a position corresponding to P14 of SEQ ID NO:
  • the invention provides an antibody comprising (e.g., in a light chain or a fragment thereof): (i) the amino acid sequence of SEQ ID NO: 152, with an unnatural amino acid (UAA) at a position corresponding to V6 of SEQ ID NO: 152; the amino acid sequence of SEQ ID NO: 153, with an unnatural amino acid (UAA) at a position corresponding to V15 of SEQ ID NO: 153; the amino acid sequence of SEQ ID NO: 154, with an unnatural amino acid (UAA) at a position corresponding to V15 of SEQ ID NO: 154; or the amino acid sequence of SEQ ID NO: 155, with an unnatural amino acid (UAA) at a position corresponding to V15 of SEQ ID NO: 155; (ii) the amino acid sequence of SEQ ID NO: 156, with an unnatural amino acid (UAA) at a position corresponding to T6 of SEQ ID NO: 156; the amino acid sequence of SEQ ID NO
  • the UAA is: (i) a tryptophan analog (e.g., 5-HTP and 5- AzW); (ii) a leucine analog (e.g., LCA and Cys-5-N3); (iii) a tyrosine analog (e.g., OmeY, AzF, and OpropY); or (iv) a pyrrolysine analog (e.g., BocK, CpK, and AzK).
  • a tryptophan analog e.g., 5-HTP and 5- AzW
  • a leucine analog e.g., LCA and Cys-5-N3
  • a tyrosine analog e.g., OmeY, AzF, and OpropY
  • a pyrrolysine analog e.g., BocK, CpK, and AzK
  • the antibody further comprises a chemical modification of the unnatural amino acid (UAA), e.g., a conjugation to a molecule, e.g., a detectable label or a drug (e.g., a small molecule drug).
  • UAA unnatural amino acid
  • the molecule is: AEB, AEVB, AFP, an amatoxin, an auristatin (e.g., auristatin E), a calicheamicin, CC-1065 or a CC-1065 analog, chalicheamicin, combretastatin, DM1, DM4, docetaxel, dolastatin-10, DUBA, a duocarmycin, echinomycin, FAM, maytansine, a maytansinoid, MMAD, MMAE, MMAF, a morpholino-doxorubicin (e.g., cyanomorpholino-doxorubicin), netropsin, an oligonucleotide (e.g., a DNA, RNA, or LNA oligonucleotide), paclitaxel, PBD, a peptide (e.g., a therapeutic peptide), rhizoxin, a small molecule (e
  • the molecule is conjugated to the UAA by a linker, e.g., a cleavable linker, a non-cleavable linker, a peptide-based linker, or a PEG-based linker.
  • a linker e.g., a cleavable linker, a non-cleavable linker, a peptide-based linker, or a PEG-based linker.
  • the antibody has an average drug antibody ratio (DAR) of at least 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0, as measured by hydrophobic interaction chromatography (HIC).
  • HIC hydrophobic interaction chromatography
  • the antibody has an average drug antibody ratio (DAR) that is within 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the number of UAAs in the antibody.
  • DAR average drug antibody ratio
  • at least 40%, 50%, 60%, 70%, 80%, or 90% of the antibody remains following incubation in human plasma for 72 hours at 37 °C.
  • at least 40%, 50%, 60%, 70%, 80%, or 90% of the antibody remains following incubation with Cathepsin B for 240 minutes at 37 °C.
  • the antibody has a binding affinity for a target antigen of 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.75 nM, 0.5 nM, 0.1 nM, 0.075 nM, or 0.05 nM or lower, as measured by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the antibody has a binding affinity for a target antigen that is within 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 1.0 fold, 1.5 fold, 2.0 fold, 3.0 fold, 4.0 fold, 5.0 fold, 6.0 fold, 8.0 fold, or 10.0 fold of the binding affinity for the target antigen of a reference antibody, wherein the reference antibody is an otherwise identical antibody that does not comprise the UAA, as measured by ELISA.
  • the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.
  • the invention provides a method of producing any of the foregoing antibodies.
  • the method comprises culturing a cell with: (i) a nucleic acid comprising a nucleotide sequence encoding a tRNA comprising an anticodon that hybridizes to a codon selected from UAG, UGA, and UAA, and is capable of being charged with the unnatural amino acid (UAA); (ii) a nucleic acid comprising a nucleotide sequence encoding an aminoacyl-tRNA synthetase capable of charging the tRNA with the unnatural amino acid (UAA); and (iii) a nucleic acid comprising a nucleotide sequence encoding a heavy chain, a light chain, or a combination of a heavy chain and light chain of the antibody and comprising the codon selected from UAG, UGA, and UAA; under conditions that permit the tRNA, when expressed in the cell and charged with the unnatural amino acid (UAA), to hybridize to the codon and direct the incorporation of the unnatural amino acid (UAA) into the antibody
  • the amount of the antibody comprising the unnatural amino acid (UAA) expressed by the cell is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the amount of a reference antibody expressed by the same cell or a similar cell.
  • the reference antibody is an otherwise identical antibody that does not comprise the UAA, for example, the reference antibody comprises a wild-type amino acid residue at the position corresponding to the unnatural amino acid (UAA).
  • the tRNA is an analog or derivative of a prokaryotic tryptophanyl-tRNA, e.g., an E. coli tryptophanyl-tRNA.
  • the tRNA may comprise a nucleotide sequence selected from any one of SEQ ID NOs: 49-54 or 108-113.
  • the aminoacyl-tRNA synthetase is an analog or derivative of a prokaryotic tryptophanyl-tRNA synthetase, e.g., an E. coli tryptophanyl-tRNA synthetase.
  • the aminoacyl-tRNA synthetase may comprise an amino acid sequence selected from any one of SEQ ID NOs: 44-48.
  • the codon is UGA.
  • the UAA is a tryptophan analog, e.g., a non-naturally occurring tryptophan analog.
  • the UAA is 5-HTP or 5-AzW.
  • the tRNA is an analog or derivative of a prokaryotic leucyl- tRNA, e.g., an E. coli leucyl-tRNA.
  • the tRNA may comprise a nucleotide sequence selected from any one of SEQ ID NOs: 16-43.
  • the aminoacyl-tRNA synthetase is an analog or derivative of a prokaryotic leucyl-tRNA synthetase, e.g., an E. coli leucyl-tRNA synthetase.
  • the aminoacyl-tRNA synthetase may comprise an amino acid sequence selected from any one of SEQ ID NOs: 1- 15.
  • the codon is UAG.
  • the UAA is a leucine analog, e.g., a non-naturally occurring leucine analog.
  • the UAA is LCA or Cys-5-N3.
  • the tRNA is an analog or derivative of a prokaryotic tyrosyl- tRNA, e.g., an E. coli tyrosyl-tRNA.
  • the tRNA may comprise a nucleotide sequence selected from any one of SEQ ID NOs: 68-69 or 104-105.
  • the aminoacyl-tRNA synthetase is an analog or derivative of a prokaryotic tyrosyl-tRNA synthetase, e.g., an E. coli tyrosyl-tRNA synthetase.
  • the aminoacyl-tRNA synthetase may comprise the amino acid sequence of SEQ ID NO: 70.
  • the codon is UAG.
  • the UAA is a tyrosine analog, e.g., a non-naturally occurring tyrosine analog.
  • the UAA is OmeY, AzF, or OpropY.
  • the tRNA is an analog or derivative of an archael pyrrolysyl- tRNA, e.g., an M. barkeri pyrrolysyl-tRNA.
  • the tRNA may comprise a nucleotide sequence selected from any one of SEQ ID NOs: 72-100 or 106-107.
  • the aminoacyl-tRNA synthetase is an analog or derivative of an archael pyrrolysyl-tRNA synthetase, e.g., an M. barkeri pyrrolysyl-tRNA synthetase.
  • the aminoacyl-tRNA synthetase may comprise the amino acid sequence of SEQ ID NO: 101.
  • the codon is UAG.
  • the UAA is a pyrrolysine analog, e.g., a non-naturally occurring pyrrolysine analog.
  • the UAA is BocK, CpK, or AzK.
  • the cell is a human cell, e.g., a human embryonic kidney (HEK) or a Chinese hamster ovary (CHO) cell.
  • the method further comprises contacting the cell with the UAA.
  • the method further comprises purifying the antibody.
  • the method further comprises chemically modifying the UAA, for example, conjugating the UAA to a conjugation to a molecule, e.g., a detectable label or a drug (e.g., a small molecule drug).
  • a conjugation to a molecule e.g., a detectable label or a drug (e.g., a small molecule drug).
  • the molecule is: AEB, AEVB, AFP, an amatoxin, an auristatin (e.g., auristatin E), a calicheamicin, CC-1065 or a CC-1065 analog, chalicheamicin, combretastatin, DM1, DM4, docetaxel, dolastatin-10, DUBA, a duocarmycin, echinomycin, FAM, maytansine, a maytansinoid, MMAD, MMAE, MMAF, a morpholino-doxorubicin (e.g., cyanomorpholino-doxorubicin), netropsin, an oligonucleotide (e.g., a DNA, RNA, or LNA oligonucleotide), paclitaxel, PBD, a peptide (e.g., a therapeutic peptide), rhizoxin, a small molecule (e
  • the molecule is conjugated to the UAA by a linker, e.g., a cleavable linker, a non-cleavable linker, a peptide-based linker, or a PEG-based linker.
  • a linker e.g., a cleavable linker, a non-cleavable linker, a peptide-based linker, or a PEG-based linker.
  • FIGURE 2 depicts the heavy chain amino acid sequence and light chain sequence of Trastuzumab. Amino acid residues described in the present disclosure are bolded and underlined.
  • FIGURE 3 depicts an enzyme-linked immunosorbent assay (ELISA) for various antibody-drug conjugates (ADCs) conjugated to the UAA LCA.
  • FIGURE 4 depicts: [0033] Trastuzumab HC-T198—LCA antibody drug conjugate production characterization by size exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC) (FIGURE 4A), SEC and HIC with payload conjugated (FIGURE 4B and FIGURE 4C).
  • SEC size exclusion chromatography
  • HIC hydrophobic interaction chromatography
  • Trastuzumab HC-A143 LCA antibody drug conjugate production characterization by size exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC) (FIGURE 4D), SEC and HIC with payload conjugated (FIGURE 4E).
  • Trastuzumab HC-A121 LCA antibody drug conjugate production characterization by size exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC) (FIGURE 4F), SEC and HIC with payload conjugated (FIGURE 4G).
  • Trastuzumab LC-K107 LCA antibody drug conjugate production by size exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC) (FIGURE 4H), SEC and HIC with payload conjugated (FIGURE 4I).
  • Trastuzumab LC-T109 LCA antibody drug conjugate production characterization by size exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC) (FIGURE 4J), SEC and HIC with payload conjugated (FIGURE 4K).
  • FIGURE 5 depicts: [0039] Liquid chromatography–mass spectrometry (LC-MS) for Trastuzumab HC-T198- LCA, deconvoluted values shown in FIGURE 5A and FIGURE 5C, and an LC-MS chromatogram in FIGURE 5B. [0040] LC-MS for Trastuzumab HC-T198-BCN-PEG4-MMAD, deconvoluted values shown in FIGURE 5D and an LC-MS chromatogram in FIGURE 5E.
  • LC-MS Liquid chromatography–mass spectrometry
  • LC-MS for Trastuzumab HC-T198-HTP deconvoluted values shown in FIGURE 5F and FIGURE 5H, and an LC-MS chromatogram in FIGURE 5G.
  • LC-MS for Trastuzumab HC-T198-HTP + Diazonium-Biotin deconvoluted values shown in FIGURE 5I and FIGURE 5K, and an LC-MS chromatogram in FIGURE 5J.
  • LC-MS for Trastuzumab HC-A143-LCA deconvoluted values shown in FIGURE 5L and FIGURE 5N, and an LC-MS chromatogram in FIGURE 5M.
  • LC-MS for Trastuzumab HC-A143-BCN-PEG4-MMAD deconvoluted values shown in FIGURE 5O and FIGURE 5Q, and an LC-MS chromatogram in FIGURE 5P.
  • LC-MS for Trastuzumab HC-A121/LCA deconvoluted values shown in FIGURE 5R and FIGURE 5T
  • LC-MS chromatogram in FIGURE 5S LC-MS for Trastuzumab HC-A121/BCN-PEG8-MMAD deconvoluted values shown in FIGURE 5U
  • an LC-MS chromatogram in FIGURE 5V LC-MS chromatogram in FIGURE 5V.
  • FIGURE 6A depicts a subset of UAAs that are exemplary substrates for a leucyl tRNA-synthetase.
  • FIGURE 6B depicts a subset of UAAs that are exemplary substrates for a tryptophanyl tRNA-synthetase.
  • FIGURE 6C depicts a subset of UAAs that are exemplary substrates for a leucyl tRNA-synthetase.
  • FIGURE 6D depicts a subset of UAAs that are exemplary substrates for a leucyl tRNA-synthetase.
  • FIGURE 6E depicts UAAs C5Az, LCA, and AzW.
  • FIGURE 6F depicts a subset of UAAs that are exemplary substrates for a tyrosyl tRNA-synthetase.
  • FIGURE 6G depicts a subset of UAAs that are exemplary substrates for a pyrrolysyl tRNA-synthetase.
  • FIGURE 7 presents a summary of antibody studies of the present disclosure, including ADC details.
  • FIGURE 8 depicts the chemical structures of example payloads BCN-PEG4- MMAD and BCN-PEG8-MMAD.
  • FIGURE 9 depicts the chemical structures of example payloads DBCO-PEG4- Ahx-DM1, DBCO-PEG4-MMAF, and DBCO-PEG4-VC-MMAE.
  • FIGURE 10 depicts the chemical structures of example payloads DBCO-PEG4- LNA, DBCO-FAM, and DBCO-PEG8-VC-Nontoxic-small molecule (NonToxic SM).
  • FIGURE 11 depicts the chemical structures of example payloads Diazo- Conjugate MMAF and Diazo-Conjugate biotin.
  • FIGURE 12 depicts expression levels of LCA-containing antibodies at single sites of UAA incorporation.
  • FIGURE 13 presents a summary of expression levels of antibodies with single sites of LCA incorporation as described herein.
  • FIGURE 14 depicts the average drug-antibody ratio (DAR) of LCA-containing antibodies with a variety of payloads.
  • FIGURE 15 presents a summary of the average drug-antibody ratio (DAR) of LCA-containing antibodies with a variety of payloads.
  • FIGURE 16 depicts the size-exclusion chromatography (SEC) aggregation data for the LCA-based ADCs.
  • FIGURE 17 presents a summary of the size-exclusion chromatography (SEC) aggregation data for the LCA-based ADCs.
  • FIGURE 18 depicts stability in human plasma of LCA-based ADCs.
  • FIGURE 19 presents a summary of stability in human plasma for LCA-based ADCs.
  • FIGURE 20 depicts percentage cleavage of payload following incubation with cathepsin B for LCA-based ADCs.
  • FIGURE 21 presents a summary of percentage cleavage of payload following incubation with cathepsin B for LCA-based ADCs.
  • FIGURE 22A depicts binding of ADCs to HER2 as determined by ELISA.
  • FIGURE 22B presents EC50 values for the ELISA, demonstrating that sub-nanomolar affinity is maintained with ADCs of the present disclosure.
  • FIGURE 23 depicts a table of the IC50 values for ADCs containing DBCO- PEG4/PEG8-MMAD payload as determined by cytotoxicity studies using Cell-Titer Glo®.
  • FIGURE 24A and FIGURE 24B depicts cytotoxicity traces for the indicated ADCs.
  • FIGURE 25A and FIGURE 25B depict T198 cytotoxicity curves in comparison to a T198 MC-MMAD cysteine site-specific mutant.
  • FIGURE 26A and FIGURE 26B depict A143 cytotoxicity curves in comparison to a T198 MC-MMAD cysteine site-specific mutant.
  • FIGURE 27A and FIGURE 27B depict A121 curves in comparison to a T198 MC-MMAD cysteine site-specific mutant.
  • FIGURE 28A and FIGURE 28B depict T109 curves in comparison to a T198 MC-MMAD cysteine site-specific mutant
  • FIGURE 29A and FIGURE 29B show cytotoxicity comparison for PEG4 and PEG8 payloads across tested sites.
  • FIGURE 30A and FIGURE 30B show cytotoxicity comparison for PEG4 and PEG8 payloads across tested sites.
  • FIGURE 31 presents a summary of yield, aggregation, and hydrophobicity data obtained using HTP ADCs.
  • FIGURE 32 presents a summary of yield, aggregation, and hydrophobicity data obtained using ADCs with multiple UAAs (LCA and HTP) incorporated. Values for trastuzumab are presented in FIGURE 32A, and values for a non-Trastuzumab IgG1 and a bispecific antibody are presented in FIGURE 32B.
  • DETAILED DESCRIPTION [0083] The invention is based, in part, on the discovery of specified positions in antibodies that allow for efficient incorporation of one, or more, unnatural amino acids (UAAs) into the antibodies. The invention is further based, in part, on the discovery of combinations of specified positions in antibodies, UAAs for incorporation at those positions, and molecules for conjugation at those UAAs.
  • the combinations of position, UAA, and molecule allow for the efficient generation of antibody conjugates with desirable properties, including, for example, drug to antibody ratio (DAR), lack of aggregation, stability, and binding affinity.
  • DAR drug to antibody ratio
  • the invention provides an antibody including a UAA at one or more positions corresponding to P14, G66, D73, L155, A121, K124, T138, A143, V157, T158, S160, T167, T198, N204, V205, N206, K213, D215, I256, K277, Y281, K291, K293, N300, or F407 of an antibody heavy chain or heavy chain fragment (e.g., at the corresponding positions in SEQ ID NO: 121).
  • the invention provides an antibody including a UAA at one or more positions corresponding to V15, T20, R24, S60, S66, K107, T109, V110, A111, Q147, L154, G157, K169 A193, V205, T206, or S208 of an antibody light chain or light chain fragment (e.g., at the corresponding positions in SEQ ID NO: 155).
  • the invention provides a method of producing any of the foregoing antibodies.
  • the method comprises culturing a cell with: (i) a nucleic acid comprising a nucleotide sequence encoding a tRNA comprising an anticodon that hybridizes to a codon selected from UAG, UGA, and UAA, and is capable of being charged with the unnatural amino acid (UAA); (ii) a nucleic acid comprising a nucleotide sequence encoding an aminoacyl-tRNA synthetase capable of charging the tRNA with the unnatural amino acid (UAA); and (iii) a nucleic acid comprising a nucleotide sequence encoding a heavy chain, a light chain, or a combination of a heavy chain and light chain of the antibody and comprising the codon selected from UAG, UGA, and UAA; under conditions that permit the tRNA, when expressed in the cell and charged with the unnatural amino acid (UAA), to hybridize to the codon and direct the incorporation of the unnatural amino acid (UAA) into the antibody
  • orthogonal refers to a molecule (e.g., an orthogonal tRNA or an orthogonal aminoacyl-tRNA synthetase) that is used with reduced efficiency by an expression system of interest (e.g., an endogenous cellular translation system).
  • an orthogonal tRNA in a translation system of interest is aminoacylated by any endogenous aminoacyl-tRNA synthetase of the translation system of interest with reduced or even zero efficiency, when compared to aminoacylation of an endogenous tRNA by an endogenous aminoacyl-tRNA synthetase.
  • an orthogonal aminoacyl-tRNA synthetase aminoacylates any endogenous tRNA in the translation system of interest with reduced or even zero efficiency, as compared to aminoacylation of an endogenous tRNA by an endogenous aminoacyl-tRNA synthetase.
  • UAAs unnatural amino acids
  • antibody is understood to mean an intact antibody (e.g., an intact monoclonal antibody), or a fragment thereof, such as a Fc fragment of an antibody (e.g., an Fc fragment of a monoclonal antibody), or an antigen- binding fragment of an antibody (e.g., an antigen-binding fragment of a monoclonal antibody), including an intact antibody, antigen-binding fragment, or Fc fragment that has been modified, engineered, or chemically conjugated.
  • antigen-binding fragments include Fab, Fab’, (Fab’) 2 , Fv, single chain antibodies (e.g., scFv), minibodies, and diabodies.
  • An example of a chemically conjugated antibody is an antibody conjugated to a toxin moiety.
  • antibodies are multimeric proteins that contain four polypeptide chains. Two of the polypeptide chains are called immunoglobulin heavy chains (H chains), and two of the polypeptide chains are called immunoglobulin light chains (L chains). The immunoglobulin heavy and light chains are connected by an interchain disulfide bond. The immunoglobulin heavy chains are connected by interchain disulfide bonds.
  • a light chain consists of one variable region (V L ) and one constant region (C L ).
  • the heavy chain consists of one variable region (V H ) and at least three constant regions (CH 1 , CH 2 and CH 3 ).
  • the variable regions determine the binding specificity of the antibody.
  • the variable heavy (VH) and variable light (VL) regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (FR).
  • CDR complementarity determining regions
  • Human antibodies have three VH CDRs and three VL CDRs, separated by framework regions FR1-FR4. The extent of the FRs and CDRs has been defined (Kabat, E.A., et al.
  • Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • An antibody molecule may have (i) a heavy chain constant region chosen from, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly, chosen from, e.g., the (e.g., human) heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4, and/or (ii) a light chain constant region chosen from, e.g., the (e.g., human) light chain constant regions of kappa or lambda.
  • a heavy chain constant region chosen from, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly, chosen from, e.g., the (e.g., human) heavy chain constant regions of IgG1, IgG2, IgG3, and Ig
  • an unnatural amino acid can be done for a variety of purposes, including tailoring changes in antibody structure and/or function, changing size, acidity, nucleophilicity, hydrogen bonding, hydrophobicity, accessibility of protease target sites, targeting to a moiety (e.g., for a protein array), adding a biologically active molecule, attaching a polymer, attaching a radionuclide, modulating serum half-life, modulating tissue penetration (e.g., tumors), modulating active transport, modulating tissue, cell or organ specificity or distribution, modulating immunogenicity, modulating protease resistance, etc.
  • Antibodies that include an unnatural amino acid can have enhanced or even entirely new biophysical properties.
  • an unnatural amino acid for example, the following properties are optionally modified by inclusion of an unnatural amino acid into antibody: toxicity, biodistribution, structural properties, spectroscopic properties, chemical and/or photochemical properties, catalytic ability, half-life (including but not limited to, serum half-life), ability to react with other molecules, including but not limited to, covalently or noncovalently, and the like.
  • the compositions including antibodies that include at least one unnatural amino acid are useful for, including but not limited to, novel therapeutics, diagnostics, and binding proteins.
  • An antibody may have at least one, for example, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten or more UAAs.
  • the UAAs can be the same or different. For example, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different sites in the antibody that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different UAAs.
  • An antibody may have at least one, but fewer than all, of a particular amino acid present in the antibody substituted with the UAA.
  • the UAA can be identical or different (for example, the antibody can include two or more different types of UAAs, or can include two of the same UAA).
  • the UAAs can be the same, different or a combination of a multiple unnatural amino acid of the same kind with at least one different UAA.
  • Antibodies contemplated herein may comprise a UAA in a heavy chain or a fragment thereof, for example, in one or more of a heavy chain FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, or constant region (e.g., an IgG1 constant region).
  • antibodies contemplated herein may comprise a UAA in a light chain or a fragment thereof, for example, in a light chain FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, or constant region (e.g., a kappa constant region).
  • a UAA is located in a heavy chain FR1 region of an antibody.
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 118, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 118, with an unnatural amino acid (UAA) at a position corresponding to P6 of SEQ ID NO: 118; the amino acid sequence of SEQ ID NO: 119, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 119, with an unnatural amino acid (UAA) at a position corresponding to P14 of SEQ ID NO: 119; the amino acid sequence of SEQ ID NO: 120, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 120, with an unnatural amino acid (UAA) at a position corresponding to P14 of SEQ ID NO: 120; or the amino acid sequence of
  • a UAA is located in a heavy chain CDRH2 region of an antibody.
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 122, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 122, with an unnatural amino acid (UAA) at a position corresponding to G6 of SEQ ID NO: 122; the amino acid sequence of SEQ ID NO: 123, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 123, with an unnatural amino acid (UAA) at a position corresponding to G17 of SEQ ID NO: 123; the amino acid sequence of SEQ ID NO: 120, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 120, with an unnatural
  • a UAA is located in a heavy chain FR3 region of an antibody.
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 124, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 124, with an unnatural amino acid (UAA) at a position corresponding to D6 of SEQ ID NO: 124; the amino acid sequence of SEQ ID NO: 125, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 125, with an unnatural amino acid (UAA) at a position corresponding to D7 of SEQ ID NO: 125; the amino acid sequence of SEQ ID NO: 120, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 120, with an unnatural amino
  • a UAA is located in a heavy chain FR4 region of an antibody.
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 126, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 126, with an unnatural amino acid (UAA) at a position corresponding to L6 of SEQ ID NO: 126; the amino acid sequence of SEQ ID NO: 127, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 127, with an unnatural amino acid (UAA) at a position corresponding to L6 of SEQ ID NO: 127; the amino acid sequence of SEQ ID NO: 120, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 120, with an unnatural amino
  • a UAA is located in a heavy chain CH1 region of an antibody.
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 128, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 128, with an unnatural amino acid (UAA) at a position corresponding to A6 of SEQ ID NO: 128; the amino acid sequence of SEQ ID NO: 185, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 185, with an unnatural amino acid (UAA) at a position corresponding to A6 of SEQ ID NO: 185; the amino acid sequence of SEQ ID NO: 129, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 129, with an unnatural amino acid
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 131, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 131, with an unnatural amino acid (UAA) at a position corresponding to K6 of SEQ ID NO: 131; the amino acid sequence of SEQ ID NO: 129, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 129, with an unnatural amino acid (UAA) at a position corresponding to K3 of SEQ ID NO: 129; the amino acid sequence of SEQ ID NO: 130, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 130, with an unnatural amino acid (UAA) at a position corresponding to K3 of SEQ ID NO: 130; or the amino acid sequence of SEQ ID NO: 130,
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 191, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 191, with an unnatural amino acid (UAA) at a position corresponding to K6 of SEQ ID NO: 191; the amino acid sequence of SEQ ID NO: 129, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 129, with an unnatural amino acid (UAA) at a position corresponding to K16 of SEQ ID NO: 129; the amino acid sequence of SEQ ID NO: 130, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 130, with an unnatural amino acid (UAA) at a position corresponding to K16 of SEQ ID NO: 130; or the amino acid sequence of SEQ ID NO:
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 132, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 132, with an unnatural amino acid (UAA) at a position corresponding to T6 of SEQ ID NO: 132; the amino acid sequence of SEQ ID NO: 186, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 186, with an unnatural amino acid (UAA) at a position corresponding to T4 of SEQ ID NO: 186; the amino acid sequence of SEQ ID NO: 129, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 129, with an unnatural amino acid (UAA) at a position corresponding to T18 of SEQ ID NO: 129; the amino acid sequence of SEQ ID NO:
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 133, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 133, with an unnatural amino acid (UAA) at a position corresponding to A6 of SEQ ID NO: 133; the amino acid sequence of SEQ ID NO: 129, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 129, with an unnatural amino acid (UAA) at a position corresponding to A23 of SEQ ID NO: 129; the amino acid sequence of SEQ ID NO: 130, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 130, with an unnatural amino acid (UAA) at a position corresponding to A23 of SEQ ID NO: 130; or the amino acid sequence of SEQ ID NO: 130,
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 134, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 134, with an unnatural amino acid (UAA) at a position corresponding to V6 of SEQ ID NO: 134; the amino acid sequence of SEQ ID NO: 129, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 129, with an unnatural amino acid (UAA) at a position corresponding to V37 of SEQ ID NO: 129; the amino acid sequence of SEQ ID NO: 130, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 130, with an unnatural amino acid (UAA) at a position corresponding to V37 of SEQ ID NO: 130; or the amino acid sequence of SEQ ID NO: 130,
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 174, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 174, with an unnatural amino acid (UAA) at a position corresponding to T6 of SEQ ID NO: 174; the amino acid sequence of SEQ ID NO: 187, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 187, with an unnatural amino acid (UAA) at a position corresponding to T5 of SEQ ID NO: 187; the amino acid sequence of SEQ ID NO: 129, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 129, with an unnatural amino acid (UAA) at a position corresponding to T38 of SEQ ID NO: 129; the amino acid sequence of SEQ ID NO:
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 135, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 135, with an unnatural amino acid (UAA) at a position corresponding to S6 of SEQ ID NO: 135; the amino acid sequence of SEQ ID NO: 187, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 187, with an unnatural amino acid (UAA) at a position corresponding to S7 of SEQ ID NO: 187; the amino acid sequence of SEQ ID NO: 129, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 129, with an unnatural amino acid (UAA) at a position corresponding to S40 of SEQ ID NO: 129; the amino acid sequence of SEQ ID NO: 135,
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 136, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 136, with an unnatural amino acid (UAA) at a position corresponding to T6 of SEQ ID NO: 136; the amino acid sequence of SEQ ID NO: 188, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 188, with an unnatural amino acid (UAA) at a position corresponding to T4 of SEQ ID NO: 188; the amino acid sequence of SEQ ID NO: 129, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 129, with an unnatural amino acid (UAA) at a position corresponding to T47 of SEQ ID NO: 129; the amino acid sequence of SEQ ID NO:
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 137, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 137, with an unnatural amino acid (UAA) at a position corresponding to T6 of SEQ ID NO: 137; the amino acid sequence of SEQ ID NO: 129, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 129, with an unnatural amino acid (UAA) at a position corresponding to T78 of SEQ ID NO: 129; the amino acid sequence of SEQ ID NO: 130, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 130, with an unnatural amino acid (UAA) at a position corresponding to T78 of SEQ ID NO: 130; or the amino acid sequence of SEQ ID NO: 130,
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 138, with an unnatural amino acid (UAA) at a position corresponding to N6 of SEQ ID NO: 138; the amino acid sequence of SEQ ID NO: 189, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 189, with an unnatural amino acid (UAA) at a position corresponding to N6 of SEQ ID NO: 189; the amino acid sequence of SEQ ID NO: 129, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 129, with an unnatural amino acid (UAA) at a position corresponding to N84 of SEQ ID NO: 129; the amino acid sequence of SEQ ID NO:
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 139, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 139, with an unnatural amino acid (UAA) at a position corresponding to V6 of SEQ ID NO: 139; the amino acid sequence of SEQ ID NO: 190, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 190, with an unnatural amino acid (UAA) at a position corresponding to V6 of SEQ ID NO: 190; the amino acid sequence of SEQ ID NO: 129, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 129, with an unnatural amino acid (UAA) at a position corresponding to V85 of SEQ ID NO: 129; the amino acid sequence of SEQ ID NO:
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 140, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 140, with an unnatural amino acid (UAA) at a position corresponding to N6 of SEQ ID NO: 140; the amino acid sequence of SEQ ID NO: 189, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 189, with an unnatural amino acid (UAA) at a position corresponding to N8 of SEQ ID NO: 189; the amino acid sequence of SEQ ID NO: 129, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 129, with an unnatural amino acid (UAA) at a position corresponding to N86 of SEQ ID NO: 129; the amino acid sequence of SEQ ID NO: 140, or
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 141, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 141, with an unnatural amino acid (UAA) at a position corresponding to K6 of SEQ ID NO: 141; the amino acid sequence of SEQ ID NO: 192, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 192, with an unnatural amino acid (UAA) at a position corresponding to K5 of SEQ ID NO: 192; the amino acid sequence of SEQ ID NO: 129, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 129, with an unnatural amino acid (UAA) at a position corresponding to K93 of SEQ ID NO: 129; the amino acid sequence of SEQ ID NO:
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 142, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 142, with an unnatural amino acid (UAA) at a position corresponding to D6 of SEQ ID NO: 142; the amino acid sequence of SEQ ID NO: 129, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 129, with an unnatural amino acid (UAA) at a position corresponding to D95 of SEQ ID NO: 129; the amino acid sequence of SEQ ID NO: 130, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 130, with an unnatural amino acid (UAA) at a position corresponding to D95 of SEQ ID NO: 130; or the amino acid sequence of SEQ ID NO: 130,
  • a UAA is located in a heavy chain CH2 region of an antibody.
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 143, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 143, with an unnatural amino acid (UAA) at a position corresponding to I6 of SEQ ID NO: 143; the amino acid sequence of SEQ ID NO: 144, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 144, with an unnatural amino acid (UAA) at a position corresponding to I23 of SEQ ID NO: 144; the amino acid sequence of SEQ ID NO: 130, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 130, with an unnatural amino acid
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 145, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 145, with an unnatural amino acid (UAA) at a position corresponding to K6 of SEQ ID NO: 145; the amino acid sequence of SEQ ID NO: 193, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 193, with an unnatural amino acid (UAA) at a position corresponding to K6 of SEQ ID NO: 193; the amino acid sequence of SEQ ID NO: 144, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 144, with an unnatural amino acid (UAA) at a position corresponding to K44 of SEQ ID NO: 144; the amino acid sequence of SEQ ID NO: 145
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 146, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 146, with an unnatural amino acid (UAA) at a position corresponding to Y6 of SEQ ID NO: 146; the amino acid sequence of SEQ ID NO: 193, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 193, with an unnatural amino acid (UAA) at a position corresponding to Y10 of SEQ ID NO: 193; the amino acid sequence of SEQ ID NO: 144, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 144, with an unnatural amino acid (UAA) at a position corresponding to Y48 of SEQ ID NO: 144; the amino acid sequence of
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 147, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 147, with an unnatural amino acid (UAA) at a position corresponding to K6 of SEQ ID NO: 147; the amino acid sequence of SEQ ID NO: 144, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 144, with an unnatural amino acid (UAA) at a position corresponding to K58 of SEQ ID NO: 144; the amino acid sequence of SEQ ID NO: 130, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 130, with an unnatural amino acid (UAA) at a position corresponding to K171 of SEQ ID NO: 130; or the amino acid sequence of SEQ ID NO:
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 148, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 148, with an unnatural amino acid (UAA) at a position corresponding to K6 of SEQ ID NO: 148; the amino acid sequence of SEQ ID NO: 194, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 194, with an unnatural amino acid (UAA) at a position corresponding to K7 of SEQ ID NO: 194; the amino acid sequence of SEQ ID NO: 144, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 144, with an unnatural amino acid (UAA) at a position corresponding to K60 of SEQ ID NO: 144; the amino acid sequence of SEQ ID NO: 148
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 149, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 149, with an unnatural amino acid (UAA) at a position corresponding to N6 of SEQ ID NO: 149; the amino acid sequence of SEQ ID NO: 144, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 144, with an unnatural amino acid (UAA) at a position corresponding to N67 of SEQ ID NO: 144; the amino acid sequence of SEQ ID NO: 130, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 130, with an unnatural amino acid (UAA) at a position corresponding to N180 of SEQ ID NO: 130; or the amino acid sequence of SEQ ID NO: 149
  • a UAA is located in a heavy chain CH3 region of an antibody.
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 150, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 150, with an unnatural amino acid (UAA) at a position corresponding to F6 of SEQ ID NO: 150; the amino acid sequence of SEQ ID NO: 151, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 151, with an unnatural amino acid (UAA) at a position corresponding to F64 of SEQ ID NO: 151; the amino acid sequence of SEQ ID NO: 130, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 130, with an unnatural amino acid (UAA
  • a UAA is located in a light chain FR1 region of an antibody.
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 152, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 152, with an unnatural amino acid (UAA) at a position corresponding to V6 of SEQ ID NO: 152; the amino acid sequence of SEQ ID NO: 153, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 153, with an unnatural amino acid (UAA) at a position corresponding to V15 of SEQ ID NO: 153; the amino acid sequence of SEQ ID NO: 154, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 154, with an unnatural amino acid (UAA) at a position corresponding to
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 156, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 156, with an unnatural amino acid (UAA) at a position corresponding to T6 of SEQ ID NO: 156; the amino acid sequence of SEQ ID NO: 153, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 153, with an unnatural amino acid (UAA) at a position corresponding to T20 of SEQ ID NO: 153; the amino acid sequence of SEQ ID NO: 154, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 154, with an unnatural amino acid (UAA) at a position corresponding to T20 of SEQ ID NO: 154; or the amino acid sequence of
  • a UAA is located in a light chain CDR1 region of an antibody.
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 157, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 157, with an unnatural amino acid (UAA) at a position corresponding to R6 of SEQ ID NO: 157; the amino acid sequence of SEQ ID NO: 178, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 178, with an unnatural amino acid (UAA) at a position corresponding to R9 of SEQ ID NO: 178; the amino acid sequence of SEQ ID NO: 158, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 158, with an unnatural amino acid (UAA) at a position corresponding to
  • a UAA is located in a light chain FR3 region of an antibody.
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 159, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 159, with an unnatural amino acid (UAA) at a position corresponding to S6 of SEQ ID NO: 159; the amino acid sequence of SEQ ID NO: 159, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 159, with an unnatural amino acid (UAA) at a position corresponding to S11 of SEQ ID NO: 159; the amino acid sequence of SEQ ID NO: 179, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 179, with an unnatural amino acid (UAA) at a position corresponding to
  • a UAA is located in a light chain FR4 region of an antibody.
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 161, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 161, with an unnatural amino acid (UAA) at a position corresponding to K6 of SEQ ID NO: 161; the amino acid sequence of SEQ ID NO: 162, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 162, with an unnatural amino acid (UAA) at a position corresponding to K10 of SEQ ID NO: 162; the amino acid sequence of SEQ ID NO: 154, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 154, with an unnatural amino acid (
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 163, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 163, with an unnatural amino acid (UAA) at a position corresponding to T6 of SEQ ID NO: 163; the amino acid sequence of SEQ ID NO: 162, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 162, with an unnatural amino acid (UAA) at a position corresponding to T12 of SEQ ID NO: 162; the amino acid sequence of SEQ ID NO: 154, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 154, with an unnatural amino acid (UAA) at a position corresponding to T109 of SEQ ID NO: 154; or the amino acid sequence of SEQ ID NO:
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 164, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 164, with an unnatural amino acid (UAA) at a position corresponding to V6 of SEQ ID NO: 164; the amino acid sequence of SEQ ID NO: 162, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 162, with an unnatural amino acid (UAA) at a position corresponding to V13 of SEQ ID NO: 162; the amino acid sequence of SEQ ID NO: 154, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 154, with an unnatural amino acid (UAA) at a position corresponding to V110 of SEQ ID NO: 154; or the amino acid sequence of SEQ ID NO:
  • a UAA is located in a light chain constant region of an antibody.
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 165, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 165, with an unnatural amino acid (UAA) at a position corresponding to A6 of SEQ ID NO: 165; the amino acid sequence of SEQ ID NO: 166, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 165, with an unnatural amino acid (UAA) at a position corresponding to A1 of SEQ ID NO: 166; or the amino acid sequence of SEQ ID NO: 155, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 155, with an unnatural
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 167, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 167, with an unnatural amino acid (UAA) at a position corresponding to Q6 of SEQ ID NO: 167; the amino acid sequence of SEQ ID NO: 180, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 180, with an unnatural amino acid (UAA) at a position corresponding to Q7 of SEQ ID NO: 180; the amino acid sequence of SEQ ID NO: 166, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 166, with an unnatural amino acid (UAA) at a position corresponding to Q37 of SEQ ID NO: 166; or the amino acid sequence of SEQ ID NO: 167
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 168, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 168, with an unnatural amino acid (UAA) at a position corresponding to L6 of SEQ ID NO: 168; the amino acid sequence of SEQ ID NO: 181, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 181, with an unnatural amino acid (UAA) at a position corresponding to L6 of SEQ ID NO: 181; the amino acid sequence of SEQ ID NO: 166, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 166, with an unnatural amino acid (UAA) at a position corresponding to L44 of SEQ ID NO: 166; or the amino acid sequence of SEQ ID NO:
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 169, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 169, with an unnatural amino acid (UAA) at a position corresponding to G6 of SEQ ID NO: 169; the amino acid sequence of SEQ ID NO: 166, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 166, with an unnatural amino acid (UAA) at a position corresponding to G47 of SEQ ID NO: 166; or the amino acid sequence of SEQ ID NO: 155, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 155, with an unnatural amino acid (UAA) at a position corresponding to G157 of SEQ ID NO: 155.
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 175, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 175, with an unnatural amino acid (UAA) at a position corresponding to K6 of SEQ ID NO: 175; the amino acid sequence of SEQ ID NO: 166, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 166, with an unnatural amino acid (UAA) at a position corresponding to K59 of SEQ ID NO: 166; or the amino acid sequence of SEQ ID NO: 155, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 155, with an unnatural amino acid (UAA) at a position corresponding to K169 of SEQ ID NO: 155.
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 170, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 170, with an unnatural amino acid (UAA) at a position corresponding to A6 of SEQ ID NO: 170; the amino acid sequence of SEQ ID NO: 182, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 182, with an unnatural amino acid (UAA) at a position corresponding to A6 of SEQ ID NO: 182; the amino acid sequence of SEQ ID NO: 166, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 166, with an unnatural amino acid (UAA) at a position corresponding to A83 of SEQ ID NO: 166; or the amino acid sequence of SEQ ID NO: 1
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 171, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 171, with an unnatural amino acid (UAA) at a position corresponding to V6 of SEQ ID NO: 171; the amino acid sequence of SEQ ID NO: 183, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 183, with an unnatural amino acid (UAA) at a position corresponding to V5 of SEQ ID NO: 183; the amino acid sequence of SEQ ID NO: 166, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 166, with an unnatural amino acid (UAA) at a position corresponding to V95 of SEQ ID NO: 166; or the amino acid sequence of SEQ ID NO: 17
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 172, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 172, with an unnatural amino acid (UAA) at a position corresponding to T6 of SEQ ID NO: 172; the amino acid sequence of SEQ ID NO: 184, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 184, with an unnatural amino acid (UAA) at a position corresponding to T5 of SEQ ID NO: 184; the amino acid sequence of SEQ ID NO: 166, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 166, with an unnatural amino acid (UAA) at a position corresponding to T96 of SEQ ID NO: 166; or the amino acid sequence of
  • the antibody comprises: the amino acid sequence of SEQ ID NO: 173, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 173, with an unnatural amino acid (UAA) at a position corresponding to S6 of SEQ ID NO: 173; the amino acid sequence of SEQ ID NO: 184, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 184, with an unnatural amino acid (UAA) at a position corresponding to S7 of SEQ ID NO: 184; the amino acid sequence of SEQ ID NO: 166, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 166, with an unnatural amino acid (UAA) at a position corresponding to S98 of SEQ ID NO: 166; or the amino acid sequence of SEQ ID NO: 17
  • first position in a first antibody, antibody fragment, or amino acid sequence is considered to “correspond” with a second position in a second, different antibody, antibody fragment, or amino acid sequence, if a person of skill in the art would understand the first and second positions to correspond to the same position in the primary, secondary, or tertiary structure of their respective antibody, antibody fragment, or amino acid sequence. It is understood that the first and second positions may correspond to each other even if they have a different numbered position relative to N-terminus of their respective antibody, antibody fragment, or amino acid sequence, or if a different amino acid is present at the first and second positions.
  • BLAST Basic, secondary, or tertiary structure analysis of antibodies, antibody fragments, or amino acid sequences
  • Sequence identity may be determined in various ways that are within the skill of a person skilled in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
  • BLAST Basic Local Alignment Search Tool
  • analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al., (1990) PROC. NATL. ACAD. SCI.
  • the search parameters for histogram, descriptions, alignments, expect i.e., the statistical significance threshold for reporting matches against database sequences
  • cutoff, matrix and filter are at the default settings.
  • the default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al., (1992) PROC. NATL. ACAD. SCI. USA 89:10915-10919, fully incorporated by reference herein).
  • the antibody comprises a first UAA at a first position which is indicated in a cell in TABLE 1, and a second UAA at a second position which is indicated in the same cell in TABLE 1.
  • antibody includes variants having one or more mutations (e.g., amino acid substitutions, deletions, or insertions) relative to a wild-type antibody sequence or an antibody sequence disclosed herein.
  • an antibody variant may comprise, consist, or consist essentially of, a single mutation, or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more than 15 mutations relative to a wild-type antibody sequence or an antibody sequence disclosed herein.
  • an antibody variant may comprise, consist, or consist essentially 1-15, 1-10, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-15, 2-10, 2-7, 2-6, 2-5, 2-4, 2-3, 3-15, 3-10, 3-7, 3-6, 3-5, or 4-10, 4-7, 4-6, 4-5, 5-10, 5-7, 5-6, 6-10, 6-7, 7-10, 7-8, or 8-10 mutations relative to a wild-type antibody sequence or an antibody sequence disclosed herein.
  • An antibody variant may comprise a conservative substitution relative to a wild-type sequence or a sequence disclosed herein.
  • the term “conservative substitution” refers to a substitution with a structurally similar amino acid.
  • conservative substitutions may include those within the following groups: Ser and Cys; Leu, Ile, and Val; Glu and Asp; Lys and Arg; Phe, Tyr, and Trp; and Gln, Asn, Glu, Asp, and His.
  • Conservative substitutions may also be defined by the BLAST (Basic Local Alignment Search Tool) algorithm, the BLOSUM substitution matrix (e.g., BLOSUM 62 matrix), or the PAM substitution:p matrix (e.g., the PAM 250 matrix).
  • the antibody may be selected from, or may be derived from an antibody selected from, adecatumumab, ascrinvacumab, cixutumumab, conatumumab, daratumumab, drozitumab, duligotumab, durvalumab, dusigitumab, enfortumab, enoticumab, epratuxumab, figitumumab, ganitumab, glembatumumab, intetumumab, ipilimumab, iratumumab, icrucumab, lexatumumab, lucatumumab, mapatumumab, narnatumab, necitumumab, nesvacumab, ofatumumab, olaratumab, panitumumab, patritumumab, pritumumab, radret
  • the antibody may bind an antigen selected from, for example, adenosine A2a receptor (A2aR), A kinase anchor protein 4 (AKAP4), B melanoma antigen (BAGE), brother of the regulator of imprinted sites (BORIS), breakpoint cluster region Abelson tyrosine kinase (BCR/ABL), CA125, CAIX, CD19, CD20, CD22, CD30, CD33, CD52, CD73, CD137, carcinoembryonic antigen (CEA), a claudin (e.g.
  • Additional exemplary cancer antigens include those found on cancer stem cells, e.g., SSEA3, SSEA4, TRA-1-60, TRA-1-81, SSEA1, CD133 (AC133), CD90 (Thy-1), CD326 (EpCAM), Cripto-1 (TDGF1), PODXL-1 (Podocalyxin-like protein 1), ABCG2, CD24, CD49f (Integrin ⁇ 6), Notch2, CD146 (MCAM), CD10 (Neprilysin), CD117 (c-KIT), CD26 (DPP-4), CXCR4, CD34, CD271, CD13 (Alanine aminopeptidase), CD56 (NCAM), CD105 (Endoglin), LGR5, CD114 (CSF3R), CD54 (ICAM-1), CXCR1, 2, TIM-3 (HAVCR2), CD55 (DAF), DLL4 (Delta-like ligand 4), CD20 (MS4A1), and CD96.
  • TABLE 2 shows antibodies and antibody-drug conjugates suitable for use in accordance with the present invention, the antigen bound by the antibody or antibody-drug conjugate, and for certain antibodies, the type of cancer targeted by the antibody or antibody- drug conjugate.
  • the antibody is, or is derived from, trastuzumab (e.g., comprising a heavy chain amino acid sequence of SEQ ID NO: 121 and a light chain amino acid sequence of SEQ ID NO: 155).
  • the antibody has a binding affinity (K D) for a target antigen of at least 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.75 nM, 0.5 nM, 0.1 nM, 0.075 nM, or 0.05 nM or lower, as measured using standard binding assays, for example, ELISA (e.g., as described in Example 3 herein), surface plasmon resonance or bio-layer interferometry.
  • K D binding affinity for a target antigen of at least 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.75 nM, 0.5 nM, 0.1 nM, 0.075
  • the antibody binds a target antigen with a K D of from about 20 nM to about 0.05 nM, from about 20 nM to about 0.075 nM, from about 20 nM to about 0.1 nM, from about 20 nM to about 0.5 nM, from about 20 nM to about 1 nM, from about 10 nM to about 0.05 nM, from about 10 nM to about 0.075 nM, from about 10 nM to about 0.1 nM, from about 10 nM to about 0.5 nM, from about 10 nM to about 1 nM, from about 5 nM to about 0.05 nM, from about 5 nM to about 0.075 nM, from about 5 nM to about 0.1 nM, from about 5 nM to about 0.5 nM, from about 5 nM to about 1 nM, from about 3 nM to about 0.05 nM, from about 3 nM to about 0.0 nM to
  • the antibody has a binding affinity (K D) for a target antigen that is within 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 1.0 fold, 1.5 fold, 2.0 fold, 3.0 fold, 4.0 fold, 5.0 fold, 6.0 fold, 8.0 fold, or 10.0 fold of the binding affinity for the target antigen of a reference antibody, wherein the reference antibody is an otherwise identical antibody that does not comprise the UAA, as measured using standard binding assays, for example, ELISA, surface plasmon resonance or bio-layer interferometry.
  • K D binding affinity for a target antigen that is within 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 1.0 fold, 1.5 fold, 2.0 fold, 3.0 fold, 4.0 fold, 5.0 fold, 6.0 fold, 8.0 fold, or 10.0 fold of the binding affinity for the target antigen of a reference antibody, wherein the reference antibody is an otherwise identical antibody that does not comprise the UAA, as measured using standard binding assays, for example, ELISA
  • the antibody may have comparable or even improved stability relative a reference antibody, wherein the reference antibody is an otherwise identical antibody that does not comprise the UAA or a molecule conjugated to the UAA.
  • the reference antibody is an otherwise identical antibody that does not comprise the UAA or a molecule conjugated to the UAA.
  • at least 40%, 50%, 60%, 70%, 80%, or 90% of the antibody remains following incubation in human plasma for 72 hours at 37 °C.
  • at least 40%, 50%, 60%, 70%, 80%, or 90% of the antibody remains following incubation with Cathepsin B for 240 minutes at 37 °C.
  • Cathepsin B stability may be assayed as described in Example 3 herein, including, for example, in 25 mM sodium acetate and 1 mM EDTA at pH 5.0.
  • the antibody has off-target binding or activity that is within 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 1.0 fold, 1.5 fold, 2.0 fold, 3.0 fold, 4.0 fold, 5.0 fold, 6.0 fold, 8.0 fold, or 10.0 fold of the off-target binding or activity of a reference antibody, wherein the reference antibody is an otherwise identical antibody that does not comprise any UAA, does not comprise the same UAA, does not comprise the same UAA at the same position, and/or does not comprise the same molecule conjugated to the UAA/antibody.
  • the antibody has off-target binding or activity that is 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 1.0 fold, 1.5 fold, 2.0 fold, 3.0 fold, 4.0 fold, 5.0 fold, 6.0 fold, 8.0 fold, or 10.0 fold less than the off-target binding or activity of a reference antibody, wherein the reference antibody is an otherwise identical antibody that does not comprise any UAA, does not comprise the same UAA, does not comprise the same UAA at the same position, and/or does not comprise the same molecule conjugated to the UAA/antibody.
  • the antibody has an efficacy or therapeutic activity (e.g., IC50) that is within 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 1.0 fold, 1.5 fold, 2.0 fold, 3.0 fold, 4.0 fold, 5.0 fold, 6.0 fold, 8.0 fold, or 10.0 fold of the efficacy or therapeutic activity of a reference antibody, wherein the reference antibody is an otherwise identical antibody that does not comprise any UAA, does not comprise the same UAA, does not comprise the same UAA at the same position, and/or does not comprise the same molecule conjugated to the UAA/antibody.
  • IC50 efficacy or therapeutic activity
  • the antibody has an efficacy or therapeutic activity (e.g., IC50) that is 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 1.0 fold, 1.5 fold, 2.0 fold, 3.0 fold, 4.0 fold, 5.0 fold, 6.0 fold, 8.0 fold, or 10.0 fold greater than the efficacy or therapeutic activity of a reference antibody, wherein the reference antibody is an otherwise identical antibody that does not comprise any UAA, does not comprise the same UAA, does not comprise the same UAA at the same position, and/or does not comprise the same molecule conjugated to the UAA/antibody.
  • IC50 efficacy or therapeutic activity
  • Efficacy or therapeutic activity may be measured by any assays known in the art, including, e.g., in vitro cytotoxicity assays as described in Example 4 herein.
  • UAAs Unnatural Amino Acids
  • the invention relates to unnatural amino acids (UAAs) and their incorporation into proteins (e.g. antibodies).
  • an unnatural amino acid refers to any amino acid, modified amino acid, or amino acid analogue other than the following twenty genetically encoded alpha-amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine. See, e.g., Biochemistry by L. Stryer, 3rd ed.1988, Freeman and Company, New York, for structures of the twenty natural amino acids.
  • unnatural amino acid also includes amino acids that occur by modification (e.g. post- translational modifications) of a natural amino acid but are not themselves naturally incorporated into a growing polypeptide chain by the translation complex.
  • unnatural amino acids typically differ from natural amino acids only in the structure of the side chain, unnatural amino acids may, for example, form amide bonds with other amino acids in the same manner in which they are formed in naturally occurring proteins. However, the unnatural amino acids have side chain groups that distinguish them from the natural amino acids.
  • the side chain may comprise an alkyl-, aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkyl, ether, thiol, seleno-, sulfonyl-, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amine, and the like, or any combination thereof.
  • Non-naturally occurring amino acids include, but are not limited to, amino acids comprising a photoactivatable cross-linker, spin-labeled amino acids, fluorescent amino acids, metal binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with novel functional groups, amino acids that covalently or noncovalently interact with other molecules, photocaged and/or photoisomerizable amino acids, amino acids comprising biotin or a biotin analogue, glycosylated amino acids such as a sugar substituted serine, other carbohydrate modified amino acids, keto-containing amino acids, amino acids comprising polyethylene glycol or polyether, heavy atom substituted amino acids, chemically cleavable and/or photocleavable amino acids, amino acids with an elongated side chains as compared to natural amino acids, including but not limited to, polyethers or long chain hydrocarbons, including but not limited to, greater than about 5 or greater than about 10 carbons, carbon-linked sugar-containing amino acids, redox-active amino acids, amino thio
  • unnatural amino acids In addition to unnatural amino acids that contain novel side chains, unnatural amino acids also optionally comprise modified backbone structures. [00125] Many unnatural amino acids are based on natural amino acids, such as tyrosine, glutamine, phenylalanine, and the like.
  • Tyrosine analogs include para-substituted tyrosines, ortho-substituted tyrosines, and meta substituted tyrosines, wherein the substituted tyrosine comprises a keto group (including but not limited to, an acetyl group), a benzoyl group, an amino group, a hydrazine, an hydroxyamine, a thiol group, a carboxy group, an isopropyl group, a methyl group, a C 6 -C 20 straight chain or branched hydrocarbon, a saturated or unsaturated hydrocarbon, an O-methyl group, a polyether group, a nitro group, or the like.
  • multiply substituted aryl rings are also contemplated.
  • Glutamine analogs include, but are not limited to, ⁇ -hydroxy derivatives, ⁇ -substituted derivatives, cyclic derivatives, and amide substituted glutamine derivatives.
  • Exemplary phenylalanine analogs include, but are not limited to, para-substituted phenylalanines, ortho-substituted phenylalanines, and meta-substituted phenylalanines, wherein the substituent comprises a hydroxy group, a methoxy group, a methyl group, an allyl group, an aldehyde, an azido, an iodo, a bromo, a keto group (including but not limited to, an acetyl group), or the like.
  • unnatural amino acids include, but are not limited to, a p-acetyl-L- phenylalanine, a p-propargyl-phenylalanine, O-methyl-L-tyrosine, an L-3-(2- naphthyl)alanine, a 3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAc ⁇ -serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L- phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L-phenylalanine, a p-benzoyl-L- phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p
  • the unnatural amino acid may be a leucine analog (also referred to herein as a derivative). In certain embodiments, the leucine analog is a non-naturally occurring leucine analog.
  • the invention provides a leucine analog depicted in FIGURE 6A, or a composition comprising the leucine analog.
  • Formula A in FIGURE 6A depicts an amino acid analog containing a side chain including a carbon containing chain n units (0-20 units) long.
  • An O, S, CH 2 , or NH is present in at position X, and another carbon containing chain of n units (0-20 units) long can follow.
  • a functional group Y is attached to the terminal carbon of second carbon containing chain (for example, functional groups 1-12 as depicted in FIGURE 6A, where R represents a linkage to the terminal carbon atom the second carbon containing side chain).
  • these functional groups can be used for bioconjugation of any amenable ligand to any protein of interest that is amenable to site- specific UAA incorporation.
  • Formula B in FIGURE 6A depicts a similar amino acid analog containing an side chains denoted as either Z-Y 2 or Z-Y 3 attached to the second carbon containing chain or the first carbon containing chain, respectively.
  • Z represents a carbon chain comprising (CH 2 )n units, where n is any integer from 0-20.
  • Y 2 or Y 3 independently, can be the same or different groups as those of Y 1 .
  • the invention also provides a leucine analog depicted in FIGURE 6C (LCA, LKET, or ACA), or a composition comprising the leucine analog depicted in FIGURE 6C.
  • Additional exemplary leucine analogs include those selected from linear alkyl halides and linear aliphatic chains comprising a functional group, for example, an alkyne, azide, cyclopropene, alkene, ketone, aldehyde, diazirine, or tetrazine functional group, as well as structures 1-6 shown in FIGURE 6D.
  • the amino and carboxylate groups both attached to the first carbon of any amino acid shown in FIGURES 6A, 6C, or 6D would constitute portions of peptide bonds when the leucine analog is incorporated into a protein or polypeptide chain.
  • the leucine analogs set forth in FIGURE 6E referred to as C5AzMe and LCA can be used in the practice of the invention. Methods for preparing leucine analogs, e.g., C5AzMe or LCA, are described in International (PCT) Publication No. WO2021026506.
  • the unnatural amino acid is a tryptophan analog (also referred to herein as a derivative).
  • the tryptophan analog is a non- naturally occurring tryptophan analog.
  • exemplary tryptophan analogs include 5- azidotryptophan, 5-propargyloxytryptophan, 5-aminotryptophan, 5-methoxytryptophan, 5-O- allyltryptophan or 5-bromotryptophan. Additional exemplary tryptophan analogs are depicted in FIGURE 6B. However, it is contemplated that the amino and carboxylate groups both attached to the first carbon of the tryptophan analogs in FIGURE 6B would constitute portions of peptide bonds when the tryptophan analog is incorporated into a protein or polypeptide chain.
  • the tryptophan analog set forth in FIGURE 6E can be used in the practice of the invention.
  • Methods for preparing tryptophan analogs, e.g., AzW, are described in International (PCT) Publication No. WO2021026506.
  • the unnatural amino acid is a tyrosine analog (also referred to herein as a derivative).
  • the tyrosine analog is a non- naturally occurring tyrosine analog.
  • Exemplary tyrosine analogs include o-methyltyrosine (OmeY), p-azidophenylalanine (AzF), o-propargyltyrosine (OpropY or PrY), and p- acetylphenylalanine (AcF).
  • Exemplary tryptophan analogs are depicted in FIGURE 6F.
  • the unnatural amino acid is a pyrrolysine analog (also referred to herein as a derivative).
  • the pyrrolysine analog is a non- naturally occurring pyrrolysine analog.
  • Exemplary pyrrolysine analogs include aminocaprylic acid (Cap), H-Lys(Boc)-OH (Boc-Lysine, BocK), azidolysine (AzK), H- propargyl-lysine (hPrK), and cyclopropenelysine (CpK).
  • Exemplary pyrrolysine analogs are depicted in FIGURE 6G.
  • Many unnatural amino acids are commercially available, e.g., from Sigma- Aldrich (St. Louis, Mo., USA), Novabiochem (Darmstadt, Germany), or Peptech (Burlington, Mass., USA).
  • the antibody comprises two or more than two UAAs
  • the antibody comprises a first unnatural amino acid (UAA) that is a tryptophan analog (e.g., a non-naturally occurring tryptophan analog) and a second UAA that is a leucine analog (e.g., a non-naturally occurring leucine analog).
  • UAA unnatural amino acid
  • the tryptophan analog is selected from 5-HTP and 5-AzW and/or the leucine analog is selected from LCA and Cys-5-N3.
  • the antibody comprises two or more than two UAAs
  • the antibody comprises a first unnatural amino acid (UAA) that is a tryptophan analog (e.g., a non-naturally occurring tryptophan analog) and a second UAA that is a tyrosine analog (e.g., a non-naturally occurring tyrosine analog).
  • UAA unnatural amino acid
  • the tryptophan analog is selected from 5-HTP and 5-AzW and/or the tyrosine analog is selected from OmeY, AzF, and OpropY UAA.
  • the antibody comprises two or more than two UAAs
  • the antibody comprises a first unnatural amino acid (UAA) that is a tryptophan analog (e.g., a non-naturally occurring tryptophan analog) and a second UAA that is a pyrrolysine analog (e.g., a non-naturally occurring pyrrolysine analog).
  • UAA unnatural amino acid
  • pyrrolysine analog e.g., a non-naturally occurring pyrrolysine analog
  • the tryptophan analog is selected from 5-HTP and 5-AzW and/or the pyrrolysine analog is selected from BocK, CpK, AzK, and CpK. III.
  • an unnatural amino acid in a polypeptide may be used to attach another molecule to the polypeptide.
  • a disclosed antibody comprises a chemical modification of an unnatural amino acid (UAA), e.g., a conjugation to a molecule.
  • UAA unnatural amino acid
  • an antibody may comprise one or more UAAs (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more than ten UAAs, each of which may be the same or different), and similarly, may be conjugated to one or more molecules (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more than ten molecules, each of which may be the same or different).
  • UAAs e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more than ten molecules, each of which may be the same or different
  • Exemplary molecules for conjugation include a label, a dye, a polymer, a water-soluble polymer, a stabilizing agent (e.g., a derivative of polyethylene glycol), a photoactivatable crosslinker, a radionuclide, a cytotoxic compound, a drug, an affinity label, a photoaffinity label, a reactive compound, a resin, a second protein or polypeptide or polypeptide analog (e.g., a therapeutic peptide or polypeptide), an antibody or antibody fragment (e.g., an anti-CD3 antibody or antibody fragment), a metal chelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA (e.g., a DNA oligonucleotide), a RNA (e.g., a DNA oligonucleotide), a LNA (e.g., a LNA oligonucleotide), an antis
  • Additional exemplary molecules for conjugation include any cytotoxic, cytostatic or immunomodulatory drug.
  • cytotoxic or immunomodulatory agents include, for example, antitubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, calmodulin inhibitors, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, maytansinoids, nitrosoureas, platinols, pore-forming compounds, purine antimetabolites, puromycins, radiation sensitizers, rapamycins, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like.
  • alkylating agents e.g., platinum complexes such as c
  • Individual cytotoxic or immunomodulatory agents include, for example, an androgen, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, buthionine sulfoximine, calicheamicin, calicheamicin derivatives, camptothecin, carboplatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin (formerly actinomycin), daunorubicin, decarbazine, DM1, DM4, docetaxel, doxorubicin, etoposide, an estrogen, 5-fluordeoxyuridine, 5-fluorouracil, gemcitabine, gramicidin D, hydroxyurea, idarubic
  • suitable cytotoxic agents include, for example, DNA minor groove binders (e.g., enediynes and lexitropsins, a CBI compound), duocarmycins, taxanes (e.g., paclitaxel and docetaxel), puromycins, vinca alkaloids, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, echinomycin, combretastatin, netropsin, epothilone A and B, estramustine, cryptophycins, cemadotin, maytansinoids, discodermolide, eleutherobin, and mitoxantrone.
  • DNA minor groove binders e.g., enediynes and lexitropsins, a CBI compound
  • duocarmycins e.g., paclitaxel and docetaxel
  • the molecule is an anti-tubulin agent.
  • anti-tubulin agents include taxanes (e.g., Taxol ® (paclitaxel), Taxotere ® (docetaxel)), T67 (Tularik) and vinca alkyloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine).
  • antitubulin agents include, for example, baccatin derivatives, taxane analogs, epothilones (e.g., epothilone A and B), nocodazole, colchicine and colcimid, estramustine, cryptophycins, cemadotin, maytansinoids, combretastatins, discodermolide, and eleutherobin.
  • the cytotoxic agent is a maytansinoid, another group of anti-tubulin agents.
  • the maytansinoid can be maytansine or DM1.
  • the molecule is an auristatin, such as auristatin E or a derivative thereof.
  • the auristatin E derivative can be an ester formed between auristatin E and a keto acid.
  • auristatin E can be reacted with paraacetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively.
  • Other typical auristatin derivatives include AFP, MMAF, and MMAE.
  • the molecule is an antimetabolite.
  • the antimetabolite can be, for example, a purine antagonist (e.g., azothioprine or mycophenolate mofetil), a dihydrofolate reductase inhibitor (e.g., methotrexate), acyclovir, ganciclovir, zidovudine, vidarabine, ribavarin, azidothymidine, cytidine arabinoside, amantadine, dideoxyuridine, iododeoxyuridine, poscarnet, or trifluridine.
  • the payload is tacrolimus, cyclosporine, FU506 or rapamycin.
  • the molecule is aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, bexarotene, bexarotene, calusterone, capecitabine, celecoxib, cladribine, Darbepoetin alfa, Denileukin diftitox, dexrazoxane, dromostanolone propionate, epirubicin, Epoetin alfa, estramustine, exemestane, Filgrastim, floxuridine, fludarabine, fulvestrant, gemcitabine, gemtuzumab ozogamicin (MYLOTARG), goserelin, idarubicin, ifosfamide, imatinib mesylate, Interferon alfa-2a, irinotecan, letrozole,
  • the molecule is an immunomodulatory agent.
  • the immunomodulatory agent can be, for example, ganciclovir, etanercept, tacrolimus, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil or methotrexate.
  • the immunomodulatory agent can be, for example, a glucocorticoid (e.g., cortisol or aldosterone) or a glucocorticoid analogue (e.g., prednisone or dexamethasone).
  • the immunomodulatory agent can be, for example, a Toll- like receptor (TLR) agonist, e.g., a TLR7 or TLR8 agonist, e.g., imiquimod, 852A, hiltonol, resiquimod, 3M-052, CpG oligodeoxynucleotides (CpG ODN), 1V270, or SD-101.
  • TLR Toll- like receptor
  • the immunomodulatory agent is an anti-inflammatory agent, such as arylcarboxylic derivatives, pyrazole-containing derivatives, oxicam derivatives and nicotinic acid derivatives.
  • Classes of anti-inflammatory agents include, for example, cyclooxygenase inhibitors, 5-lipoxygenase inhibitors, and leukotriene receptor antagonists.
  • Suitable cyclooxygenase inhibitors include meclofenamic acid, mefenamic acid, carprofen, diclofenac, diflunisal, fenbufen, fenoprofen, indomethacin, ketoprofen, nabumetone, sulindac, tenoxicam and tolmetin.
  • Leukotriene receptor antagonists include calcitriol, and ontazolast.
  • Suitable lipoxygenase inhibitors include redox inhibitors (e.g., catechol butane derivatives, nordihydroguaiaretic acid (NDGA), masoprocol, phenidone, Ianopalen, indazolinones, naphazatrom, benzofuranol, alkylhydroxylamine), and non-redox inhibitors (e.g., hydroxythiazoles, methoxyalkylthiazoles, benzopyrans and derivatives thereof, methoxytetrahydropyran, boswellic acids and acetylated derivatives of boswellic acids, and quinolinemethoxyphenylacetic acids substituted with cycloalkyl radicals), and precursors of redox inhibitors.
  • redox inhibitors e.g., catechol butane derivatives, nordihydroguaiaretic acid (NDGA), masoprocol, phenidone, Ianopalen, indazolinones, naphaz
  • lipoxygenase inhibitors include antioxidants (e.g., phenols, propyl gallate, flavonoids and/or naturally occurring substrates containing flavonoids, hydroxylated derivatives of the flavones, flavonol, dihydroquercetin, luteolin, galangin, orobol, derivatives of chalcone, 4,2',4'-trihydroxychalcone, ortho-aminophenols, N- hydroxyureas, benzofuranols, ebselen and species that increase the activity of the reducing selenoenzymes), iron chelating agents (e.g., hydroxamic acids and derivatives thereof, N- hydroxyureas, 2-benzyl-1-naphthol, catechols, hydroxylamines, carnosol trolox C, catechol, naphthol, sulfasalazine, zyleuton, 5-hydroxyanthranilic acid and 4-(omega- ary
  • lipoxygenase inhibitors include inhibitors of eicosanoids (e.g., octadecatetraenoic, eicosatetraenoic, docosapentaenoic, eicosahexaenoic and docosahexaenoic acids and esters thereof, PGE1 (prostaglandin E1), PGA2 (prostaglandin A2), viprostol, 15-monohydroxyeicosatetraenoic, 15-monohydroxy-eicosatrienoic and 15-monohydroxyeicosapentaenoic acids, and leukotrienes B5, C5 and D5), compounds interfering with calcium flows, phenothiazines, diphenylbutylamines, verapamil, fuscoside, curcumin, chlorogenic acid, caffeic acid, 5,8,11,14-eicosatetrayenoic acid (ETYA), hydroxyphenylretinamide,
  • chemotherapeutic agents include Erlotinib (TARCEVA ® , Genentech/OSI Pharm.), Bortezomib (VELCADE ® , Millennium Pharm.), Fulvestrant (FASLODEX ® , AstraZeneca), Sutent (SU11248, Pfizer), Letrozole (FEMARA ® , Novartis), Imatinib mesylate (GLEEVEC ® , Novartis), PTK787/ZK 222584 (Novartis), Oxaliplatin (Eloxatin ® , Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNE ® , Wyeth), Lapatinib (TYKERB ® , GSK572016, Glaxo Smith Kline), Lonafarnib (SCH 66336), Sorafenib (BA
  • chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN ® (cyclosphosphamide); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (e.g., bullatacin and bullatacinone); a camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (e.g., cryptophycin 1 and cryptophycin 8); dolastatin; duo
  • anti-cancer agents include aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2- pyrrolino-doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tuber
  • anti-cancer agents include anti- metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamniprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic
  • Other useful molecules include: (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE ® (megestrol acetate), AROMASIN ® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR ® (vorozole), FEMARA ® (letrozole; Nov
  • anti-angiogenic agents include MMP-2 (matrix-metalloproteinase 2) inhibitors, MMP-9 (matrix-metalloproteinase 9) inhibitors, COX-II (cyclooxygenase II) inhibitors, and VEGF receptor tyrosine kinase inhibitors.
  • VEGF receptor tyrosine kinase inhibitors include 4-(4-bromo-2- fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline (ZD6474), 4-(4- fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)-quinazoline (AZD2171), vatalanib (PTK787;) and SU11248 (sunitinib).
  • Additional exemplary molecules for conjugation include an amatoxin, chalicheamicin, DUBA, FAM, MMAD, PBD, and a toxoid.
  • an unnatural amino acid comprises a bioconjugation handle to facilitate conjugation to another molecule.
  • a method disclosed herein can be used to site-specifically incorporate two different UAAs, each with a different bioconjugation handle, into a single protein (e.g., a single antibody).
  • the two bioconjugation handles can be chosen such that they each can be chemoselectively conjugated to two different labels using mutually orthogonal conjugation chemistries.
  • Such pairs of bioconjugation handles include, for example: azide and alkyne, azide and ketone/aldehyde, azide and cyclopropene, ketone/aldehyde and cyclopropene, 5-hydroxyindole and azide, 5-hydroxyindole and cyclopropene, and 5- hydroxyindole and ketone/aldehyde.
  • the molecule can be conjugated through a variety of linking groups (linkers).
  • the linker may be a cleavable linker or a non-cleavable linker.
  • the linker may be a flexible linker or an inflexible linker.
  • the linker should be a length sufficiently long to allow the molecule and the antibody to be linked without steric hindrance from one another and sufficiently short to retain the intended activity of the antibody.
  • the linker preferably is sufficiently hydrophilic to avoid or minimize instability or insolubility of the antibody.
  • the linker should be sufficiently stable in vivo (e.g., it is not cleaved by serum, enzymes, etc.) to permit the antibody to be operative (e.g., selectively operative) in vivo.
  • the linker may be from about 1 angstroms ( ⁇ ) to about 150 ⁇ in length, or from about 1 ⁇ to about 120 ⁇ in length, or from about 5 ⁇ to about 110 ⁇ in length, or from about 10 ⁇ to about 100 ⁇ in length.
  • the linker may be greater than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 27, 30 or greater angstroms in length and/or less than about 110, 100, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, or fewer ⁇ in length.
  • the linker may be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, and 120 ⁇ in length.
  • the linker is any divalent or multivalent linker known to those of skill in the art. Generally, the linker is capable of forming covalent bonds to the molecule and the UAA.
  • Useful divalent linkers include alkylene, substituted alkylene, heteroalkylene, substituted heteroalkylene, arylene, substituted arylene, heteroarlyene and substituted heteroarylene linkers. In certain examples, the linker is C1-10 alkylene or C1-10 heteroalkylene.
  • the linker may include a water soluble polymer.
  • the water soluble polymer may be any structural form including but not limited to linear, forked or branched.
  • the water soluble polymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG), but other water soluble polymers can also be employed.
  • PEG poly(ethylene glycol)
  • PEG poly(ethylene glycol)
  • the term "PEG” is used broadly to encompass any polyethylene glycol molecule, without regard to size or to modification at an end of the PEG.
  • a PEG used in the disclosure terminates on one end with hydroxy or methoxy.
  • the PEG can terminate with a reactive group, thereby forming a bifunctional polymer.
  • Typical reactive groups can include those reactive groups that are commonly used to react with the functional groups found in the 20 common amino acids (including but not limited to, maleimide groups, activated carbonates (including but not limited to, p-nitrophenyl ester), activated esters (including but not limited to, N- hydroxysuccinimide, p-nitrophenyl ester) and aldehydes) as well as functional groups that are inert to the 20 common amino acids but that react specifically with complementary functional groups present in UAAs (including but not limited to, azide groups, and alkyne groups).
  • the functional groups found in the 20 common amino acids including but not limited to, maleimide groups, activated carbonates (including but not limited to, p-nitrophenyl ester), activated esters (including but not limited to, N- hydroxysuccinimide, p-nitrophenyl ester) and aldehydes) as well as functional groups that are inert to the 20 common amino acids but that react specifically with complementary functional groups present in UAA
  • Any molecular mass for a PEG can be used as practically desired, including but not limited to, from about 50 Daltons (Da) to 100,000 Da or more as desired (including but not limited to, sometimes 100 Da to 100,000 Da, 0.1-50 kDa, or 10-40 kDa).
  • Branched chain PEGs including but not limited to, PEG molecules with each chain having a MW ranging from 1-100 kDa (including but not limited to, 1-50 kDa or 5-20 kDa) can also be used.
  • a contemplated linker may include any appropriate number of PEG units, e.g.
  • PEG2 e.g., PEG2, PEG4, PEG6, PEG8, PEG10, PEG12, or PEG24.
  • PEG24 e.g., PEG2, PEG4, PEG6, PEG8, PEG10, PEG12, or PEG24.
  • a wide range of PEG molecules are described in, including but not limited to, the Shearwater Polymers, Inc. catalog, Nektar Therapeutics catalog. [00162] Generally, at least one terminus of the PEG molecule is available for reaction with the UAA. For example, PEG derivatives bearing alkyne and azide moieties for reaction with amino acid side chains can be used to attach PEG to UAAs as described herein.
  • the PEG will typically contain either an alkyne moiety to effect formation of the [3+2] cycloaddition product or an activated PEG species (i.e., ester, carbonate) containing a phosphine group to effect formation of the amide linkage.
  • the UAA comprises an alkyne
  • the PEG will typically contain an azide moiety to effect formation of the [3+2] Huisgen cycloaddition product.
  • the UAA comprises a tetrazine
  • the PEG will typically contain a strained alkene.
  • the PEG will typically contain a tetrazine. If the UAA comprises a carbonyl group, the PEG will typically comprise a potent nucleophile (including but not limited to, a hydrazide, hydrazine, hydroxylamine, or semicarbazide functionality) in order to effect formation of corresponding hydrazone, oxime, and semicarbazone linkages, respectively. In other alternatives, a reverse of the orientation of the reactive groups described above can be used, i.e., an azide moiety in the UAA can be reacted with a PEG derivative containing an alkyne. [00163] Many other polymers are also suitable for use in the present disclosure.
  • polymer backbones that are water-soluble, with from 2 to about 300 termini, are particularly useful.
  • suitable polymers include, but are not limited to, other poly(alkylene glycols), such as poly(propylene glycol) ("PPG"), copolymers thereof (including but not limited to copolymers of ethylene glycol and propylene glycol), terpolymers thereof, mixtures thereof, and the like.
  • PPG poly(propylene glycol)
  • the molecular weight of each chain of the polymer backbone can vary, it is typically in the range of from about 800 Da to about 100,000 Da, often from about 6,000 Da to about 80,000 Da.
  • PEG and related polymers may include degradable linkages in the polymer backbone or in the linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule.
  • ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a biologically active agent generally hydrolyze under physiological conditions to release the agent.
  • hydrolytically degradable linkages include, but are not limited to, carbonate linkages; imine linkages resulted from reaction of an amine and an aldehyde; phosphate ester linkages formed by reacting an alcohol with a phosphate group; hydrazone linkages which are reaction product of a hydrazide and an aldehyde; acetal linkages that are the reaction product of an aldehyde and an alcohol; orthoester linkages that are the reaction product of a formate and an alcohol; peptide linkages formed by an amine group, including but not limited to, at an end of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by a phosphoramidite group, including but not limited to, at the end of a polymer, and a 5' hydroxyl group of an oligonucleotide.
  • Branched linkers may be used in antibodies of the disclosure.
  • a number of different cleavable linkers are known to those of skill in the art.
  • the mechanisms for release of an agent from these linker groups include, for example, irradiation of a photolabile bond and acid-catalyzed hydrolysis.
  • the length of the linker may be predetermined or selected depending upon a desired spatial relationship between the antibody and the molecule linked to it.
  • radiodiagnostic compounds, radiotherapeutic compounds, drugs, toxins, and other agents one skilled in the art will be able to determine a suitable method for attaching a given agent to an antibody.
  • Any hetero- or homo-bifunctional linker can be used to link the conjugates.
  • the linker may have a wide range of molecular weight or molecular length. Larger or smaller molecular weight linkers may be used to provide a desired spatial relationship or conformation between the antibody and the linked entity. Linkers having longer or shorter molecular length may also be used to provide a desired space or flexibility between the antibody and the linked entity. Similarly, a linker having a particular shape or conformation may be utilized to impart a particular shape or conformation to the antibody or the linked entity, either before or after the antibody reaches its target. The functional groups present on each end of the linker may be selected to modulate the release of an antibody or a payload under desired conditions.
  • water-soluble bifunctional linkers have a dumbbell structure that includes: a) an azide, an alkyne, a hydrazine, a hydrazide, a hydroxylamine, a carbonyl, a tetrazine, or a strained alkene-containing moiety on at least a first end of a polymer backbone; and b) at least a second functional group on a second end of the polymer backbone.
  • the second functional group can be the same or different as the first functional group.
  • the second functional group in some examples, is not reactive with the first functional group.
  • water-soluble compounds that comprise at least one arm of a branched molecular structure.
  • the branched molecular structure can be dendritic.
  • Further illustrative linkers include, for example, malC, thioether, AcBut, valine-citrulline peptide, malC-valine-citrulline peptide, hydrazone, and disulfide.
  • coupling of antibody and molecule can be accomplished via a crosslinking agent.
  • crosslinking agent There are several intermolecular crosslinking agents which can be utilized, see for example, Means and Feeney, CHEMICAL MODIFICATION OF PROTEINS, Holden-Day, 1974, pp.39-43.
  • SPDP N- succinimidyl3-(2-pyridyldithio) propionate
  • N, N’- (1,3-phenylene) bismaleimide both of which are highly specific for sulfhydryl groups and form irreversible linkages
  • crosslinking agents useful for this purpose include: p,p’-difluoro-N,N’- dinitrodiphenylsulfone (which forms irreversible crosslinkages with amino and phenolic groups); dimethyl adipimidate (which is specific for amino groups); phenol-1,4- disulfonylchloride (which reacts principally with amino groups); hexamethylenediisocyanate or diisothiocyanate, or azophenyl-p-diisocyanate (which reacts principally with amino groups); glutaraldehyde (which reacts with several different side chains) and disdiazobenzidine (which reacts primarily with tyrosine and histidine); N-3- Maleimidopropanoic acid; N-6-Maleimidocaproic acid; N-11-Maleimidoundecanoic acid, 4- (N-maleimidomethyl)cyclohexane-1-carboxy-6-amidocaproic acid; 4- [
  • the crosslinking agent may be homobifunctional, i.e., having two functional groups that undergo the same reaction.
  • An example of a homobifunctional crosslinking agent is bismaleimidohexane (“BMH”).
  • BMH contains two maleimide functional groups, which react specifically with sulfhydryl-containing compounds under mild conditions (pH 6.5-7.7). The two maleimide groups are connected by a hydrocarbon chain. Therefore, BMH is useful for irreversible crosslinking of polypeptides that contain cysteine residues.
  • homobifunctional crosslinking agents include: BSOCOES (Bis(2 [Succinimidooxycarbonyloxy]ethyl) sulfone; DPDPB (1,4-Di-(3’-[2pyridyldithio]- propionamido) butane; DSS (disuccinimidyl suberate); DST (disuccinimidyl tartrate); Sulfo DST (sulfodisuccinimidyl tartrate); DSP (dithiobis(succinimidyl propionate); DTSSP (3,3’- Dithiobis(sulfosuccinimidyl propionate); EGS (ethylene glycol bis(succinimidyl succinate)); BASED (Bis( ⁇ -[4-azidosalicylamido]-ethyl)disulfide iodinatable); homobifunctional NHS crosslinking reagents (e
  • Heterobifunctional crosslinking agents have two different functional groups, for example an amine-reactive group and a thiol-reactive group, that will crosslink two moieties having free amines and thiols, respectively.
  • the most common commercially available heterobifunctional crosslinking agents have an amine reactive N- hydroxysuccinimide ester as one functional group, and a sulfhydryl reactive group as the second functional group.
  • the most common sulfhydryl reactive groups are maleimides, pyridyl disulfides and active halogens.
  • One of the functional groups can be a photoactive aryl nitrene, which upon irradiation reacts with a variety of groups.
  • heterobifunctional crosslinking agents include succinimidyl 4-(N maleimidomethyl) cyclohexane-1-carboxylate (“SMCC”), Succinimidyl-4-(N maleimidomethyl)-cyclohexane-1- carboxy(6-amidocaproate) (“LC-SMCC”), N maleimidobenzoyl-N-hydroxysuccinimide ester (“MBS”), and succinimide 4-(p-maleimidophenyl) butyrate (“SMPB”), an extended chain analog of MBS.
  • SMCC succinimidyl 4-(N maleimidomethyl) cyclohexane-1-carboxylate
  • LC-SMCC Succinimidyl-4-(N maleimidomethyl)-cyclohexane-1- carboxy(6-amidocaproate)
  • MBS N maleimidobenzoyl-N-hydroxysuccinimide ester
  • SMPB succinimide 4-(p-
  • succinimidyl group of these crosslinking agents reacts with a primary amine forming an amide bond, and the thiol-reactive maleimide forms a covalent thioether bond with the thiol group (e.g., of a cysteine).
  • Additional exemplary crosslinking agents include: BS3 ([Bis(sulfosuccinimidyl)suberate], which is a homobifunctional N-hydroxysuccinimide ester that targets accessible primary amines; NHS/EDC (N-hydroxy-succinimide and N-ethyl- ‘(dimethylaminopropyl)carbodimide, which allows for the conjugation of primary amine groups with carboxyl groups); sulfoEMCS ([N-e-Maleimido-caproic acid]hydrazide, which includes heterobifunctional reactive groups (a maleimide and an NHS-ester) that are reactive toward sulfhydryl and amino groups; hydrazide, which is useful for useful for linking carboxyl groups on exposed carbohydrates to primary amines; SATA (N-succinimidyl-S- acetylthioacetate), which is reactive towards amines and adds protected sulfhydryl groups; mono
  • crosslinking agents can be varied by the use of polymeric regions between the two reactive groups, which typically take the form of chemical linkers such as polymeric ethylene glycol or simple carbon chains, but can also include sugars, amino acids or peptides, or oligonucleotides. Polymer chain lengths of from 5 to 50 nm are typical, but can be shorter or longer as needed.
  • the crosslinking agent may comprise a ⁇ 2 carbon chain arm, a 2-5 carbon chain arm, or a 3-6 carbon chain arm.
  • Crosslinking agents often have low solubility in water. A hydrophilic moiety, such as a sulfonate group, may be added to the crosslinking agent to improve its water solubility.
  • Sulfo-MBS and sulfo-SMCC are examples of crosslinking agents modified for water solubility.
  • Many crosslinking agents yield a conjugate that is essentially non-cleavable under cellular conditions.
  • some crosslinking agents contain a covalent bond, such as a disulfide, that is cleavable under cellular conditions.
  • a disulfide such as a disulfide
  • DSP dithiobis(succinimidylpropionate)
  • SPDP N-succinimidyl 3-(2-pyridyldithio) propionate
  • Direct disulfide linkage may also be useful.
  • the linker comprises a polypeptide linker that connects or fuses the molecule to the antibody.
  • the linker may comprise hydrophilic amino acid residues, such as Gln, Ser, Gly, Glu, Pro, His and Arg.
  • the linker is a peptide containing 1-25 amino acid residues, 1-20 amino acid residues, 2-15 amino acid residues, 3-10 amino acid residues, 3-7 amino acid residues, 4- 25 amino acid residues, 4-20 amino acid residues, 4-15 amino acid residues, 4-10 amino acid residues, 5-25 amino acid residues, 5-20 amino acid residues, 5-15 amino acid residues, or 5- 10 amino acid residues.
  • Exemplary linkers include glycine and serine-rich linkers, e.g., (GlyGlyPro) n , or (GlyGlyGlyGlySer) n , where n is 1-5.
  • the linker comprises, consists, or consists essentially of GGGGS (SEQ ID NO: 176). In certain embodiments, the linker comprises, consists, or consists essentially of GGGGSGGGGS (SEQ ID NO: 177). Additional exemplary linker sequences are disclosed, e.g., in George et al. (2003) PROTEIN ENGINEERING 15:871–879, and U.S. Patent Nos.5,482,858 and 5,525,491.
  • the UAA comprises a non-natural aromatic chemical moiety (e.g., a hydroxyl-indole group; an amino-indole group; an aminophenol group; or a hydroxyl-phenol group, e.g., the UAA is 5-hydroxytryptophan (5-HTP), or an analog thereof), and/or the linker comprises a diazonium group (e.g., the linker comprises 4- nitorbenzenediazonium (4NDz); 4-carboxybenzenediazonium (4NeDz) or 4- methoxybenzenediazonium (4MCDz).
  • a non-natural aromatic chemical moiety e.g., a hydroxyl-indole group; an amino-indole group; an aminophenol group; or a hydroxyl-phenol group, e.g., the UAA is 5-hydroxytryptophan (5-HTP), or an analog thereof
  • the linker comprises a diazonium group (e.g., the linker comprises 4- nit
  • the UAA and linker may react under conditions suitable to form an azo-linkage via an azo-coupling reaction between the aromatic chemical moiety and the diazonium group.
  • DAR average drug antibody ratio
  • the antibody has an average drug antibody ratio (DAR) of at least 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0, as measured by hydrophobic interaction chromatography (HIC).
  • DAR average drug antibody ratio
  • drug antibody ratio may refer to the ratio of any conjugated molecule to antibody (e.g., a detectable label as well as a drug).
  • the antibody has an average drug antibody ratio (DAR) that is within 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the number of UAAs in the antibody.
  • DAR drug antibody ratio
  • tRNAS, aminoacyl-tRNA synthetases, and/or unnatural amino acids disclosed herein may be used to incorporate an unnatural amino acid into an antibody of interest using any appropriate translation system.
  • translation system refers to a system including components necessary to incorporate an amino acid into a growing polypeptide chain (protein).
  • Translation systems can include, e.g., ribosomes, tRNA's, synthetases, mRNA and the like.
  • Translation systems may be cellular or cell-free, and may be prokaryotic or eukaryotic.
  • translation systems may include, or be derived from, a non-eukaryotic cell, e.g., a bacterium (such as E. coli), a eukaryotic cell, e.g., a yeast cell, a mammalian cell, a plant cell, an algae cell, a fungus cell, or an insect cell.
  • a non-eukaryotic cell e.g., a bacterium (such as E. coli)
  • a eukaryotic cell e.g., a yeast cell, a mammalian cell, a plant cell, an algae cell, a fungus cell, or an insect cell.
  • Translation systems include host cells or cell lines, e.g., host cells or
  • a polypeptide of interest with an unnatural amino acid in a host cell
  • Translation systems also include whole cell preparations such as permeabilized cells or cell cultures wherein a desired nucleic acid sequence can be transcribed to mRNA and the mRNA translated. Cell-free translation systems are commercially available and many different types and systems are well-known.
  • cell-free systems include, but are not limited to, prokaryotic lysates such as Escherichia coli lysates, and eukaryotic lysates such as wheat germ extracts, insect cell lysates, rabbit reticulocyte lysates, rabbit oocyte lysates and human cell lysates.
  • Reconstituted translation systems may also be used.
  • Reconstituted translation systems may include mixtures of purified translation factors as well as combinations of lysates or lysates supplemented with purified translation factors such as initiation factor-1 (IF-1), IF-2, IF-3 ( ⁇ or ⁇ ), elongation factor T (EF-Tu), or termination factors.
  • IF-1 initiation factor-1
  • IF-2 IF-2
  • IF-3 ⁇ or ⁇
  • elongation factor T elongation factor T
  • Cell-free systems may also be coupled transcription/translation systems wherein DNA is introduced to the system, transcribed into mRNA and the mRNA is translated.
  • the invention provides methods of expressing an antibody containing an unnatural amino acid and methods of producing an antibody with one, or more, unnatural amino acids at specified positions in the antibody.
  • the methods comprise incubating a translation system (e.g., culturing or growing a host cell or cell line, e.g., a host cell or cell line disclosed herein) under conditions that permit incorporation of the unnatural amino acid into the antibody being expressed in the cell.
  • the translation system may be contacted with (e.g.
  • the cell culture medium may be contacted with) one, or more, unnatural amino acids (e.g., leucyl or tryptophanyl analogs) under conditions suitable for incorporation of the one, or more, unnatural amino acids into the antibody.
  • the antibody is expressed from a nucleic acid sequence comprising a premature stop codon.
  • the translation system may, for example, contain a leucyl-tRNA synthetase mutein (e.g., a leucyl-tRNA synthetase mutein disclosed herein) capable of charging a suppressor leucyl tRNA (e.g., a suppressor leucyl tRNA disclosed herein) with an unnatural amino acid (e.g., a leucyl analog) which is incorporated into the antibody at a position corresponding to the premature stop codon.
  • a leucyl-tRNA synthetase mutein e.g., a leucyl-tRNA synthetase mutein disclosed herein
  • an unnatural amino acid e.g., a leucyl analog
  • the leucyl suppressor tRNA comprises an anticodon sequence that hybridizes to the premature stop codon and permits the unnatural amino to be incorporated into the antibody at the position corresponding to the premature stop codon.
  • the antibody is expressed from a nucleic acid sequence comprising a premature stop codon.
  • the translation system may, for example, contain a tryptophanyl-tRNA synthetase mutein (e.g., a tryptophanyl- tRNA synthetase mutein disclosed herein) capable of charging a suppressor tryptophanyl tRNA (e.g., a suppressor tryptophanyl tRNA disclosed herein) with an unnatural amino acid (e.g., a tryptophan analog) which is incorporated into the antibody at a position corresponding to the premature stop codon.
  • a tryptophanyl-tRNA synthetase mutein e.g., a tryptophanyl- tRNA synthetase mutein disclosed herein
  • an unnatural amino acid e.g., a tryptophan analog
  • the tryptophanyl suppressor tRNA comprises an anticodon sequence that hybridizes to the premature stop codon and permits the unnatural amino to be incorporated into the antibody at the position corresponding to the premature stop codon.
  • a protein e.g., an antibody containing a UAA
  • a eukaryotic cell e.g., a mammalian cell.
  • prokaryotic cells e.g., bacteria
  • eukaryotic cells e.g., mammalian cells
  • proteins produced in mammalian cells may undergo post- translational modifications, e.g., modifications that are dependent upon enzymes located in organelles, e.g., the endoplasmic reticulum or Golgi apparatus.
  • post-translational modifications include, without limitation, sulfation, amidation, palmitation, and glycosylation (e.g., N-linked glycosylation and O-linked glycosylation).
  • a protein e.g., an antibody containing a UAA
  • the expression yield of an antibody comprising the UAA is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the expression yield of a reference antibody.
  • the amount of antibody comprising the UAA expressed by the host cell or cell line is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the amount of a reference antibody expressed by the same cell or a similar cell.
  • the reference antibody is an antibody that does not comprise the UAA but is otherwise identical to the antibody comprising the UAA.
  • the reference antibody may comprise a wild-type amino acid sequence, or comprise a wild-type amino acid residue at the position corresponding to the UAA.
  • Antibody expression may be measured by any method known in the art, including for example, Western blot or ELISA.
  • a disclosed method further comprises purifying the antibody.
  • Specific expression and purification conditions will vary depending upon the expression system employed. Purification techniques known in the art include, e.g., those employing affinity tags such as glutathione-S-transferase (GST) or histidine tags.
  • an antibody may be purified by contacting the antibody with protein A and/or protein G.
  • a disclosed method further comprises conjugating a molecule or payload to a UAA in the antibody.
  • the method comprises conjugating the molecule or payload to the UAA within 5 minutes to 48 hours at room temperature (e.g., for less than 48 hours, less than 36 hours, less than 24 hours, less than 12 hours, less than 6 hours, less than 1 hour, less than 30 minutes, less than 15 minutes, or less than 10 minutes).
  • aminoacyl-tRNA Synthetases capable of charging a tRNA with an unnatural amino acid for incorporation into a protein (e.g., an antibody).
  • aaRSs engineered aminoacyl-tRNA synthetases
  • aminoacyl-tRNA synthetase refers to any enzyme, or a functional fragment thereof, that charges, or is capable of charging, a tRNA with an amino acid (e.g., an unnatural amino acid) for incorporation into a protein.
  • the term “functional fragment” of an aminoacyl-tRNA synthetase refers to fragment of a full-length aminoacyl-tRNA synthetase that retains, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the enzymatic activity of the corresponding full-length tRNA synthetase (e.g., a naturally occurring tRNA synthetase). Aminoacyl-tRNA synthetase enzymatic activity may be assayed by any method known in the art.
  • the functional fragment comprises at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 consecutive amino acids present in a full-length tRNA synthetase (e.g., a naturally occurring aminoacyl-tRNA synthetase).
  • aminoacyl-tRNA synthetase includes variants (i.e., muteins) having one or more mutations (e.g., amino acid substitutions, deletions, or insertions) relative to a wild-type aminoacyl-tRNA synthetase sequence.
  • an aminoacyl- tRNA synthetase mutein may comprise, consist, or consist essentially of, a single mutation (e.g., a mutation contemplated herein), or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more than 15 mutations (e.g., mutations contemplated herein).
  • an aminoacyl-tRNA synthetase mutein may comprise, consist, or consist essentially 1-15, 1-10, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-15, 2-10, 2-7, 2-6, 2-5, 2-4, 2-3, 3-15, 3- 10, 3-7, 3-6, 3-5, or 4-10, 4-7, 4-6, 4-5, 5-10, 5-7, 5-6, 6-10, 6-7, 7-10, 7-8, or 8-10 mutations (e.g., mutations contemplated herein).
  • An aminoacyl-tRNA synthetase mutein may comprise a conservative substitution relative to a wild-type sequence or a sequence disclosed herein.
  • the substrate specificity of the aminoacyl-tRNA synthetase mutein is altered relative to a corresponding (or template) wild-type aminoacyl- tRNA synthetase such that only a desired unnatural amino acid, but not any of the common 20 amino acids, is charged to the substrate tRNA.
  • An aminoacyl-tRNA synthetase may be derived from a bacterial source, e.g., Escherichia coli, Thermus thermophilus, or Bacillus stearothermphilus.
  • An aminoacyl- tRNA synthetase may also be derived from an archaeal source, e.g, from the Methanosarcinacaea or Desulfitobacterium families, any of the M. barkeri (Mb), M. alvus (Ma), M. mazei (Mm) or D. hafnisense (Dh) families, Methanobacterium thermoautotrophicum, Haloferax volcanii, Halobacterium species NRC-1, or Archaeoglobus fulgidus.
  • eukaryotic sources can also be used, for example, plants, algae, protists, fungi, yeasts, or animals (e.g., mammals, insects, arthropods, etc.).
  • the terms “derivative” or “derived from” refer to a component that is isolated from or made using information from a specified molecule or organism.
  • the term “analog” refers to a component (e.g., a tRNA, tRNA synthetase, or unnatural amino acid) that is derived from or analogous with (in terms of structure and/or function) a reference component (e.g., a wild-type tRNA, a wild-type tRNA synthetase, or a natural amino acid).
  • derivatives or analogs have at least 40%, 50%, 60%, 70%, 80%, 90%, 100% or more of a given activity as a reference or originator component (e.g., wild type component).
  • a reference or originator component e.g., wild type component.
  • the aminoacyl-tRNA synthetase may aminoacylate a substrate tRNA in vitro or in vivo, and can be provided to a translation system (e.g., an in vitro translation system or a cell) as a polypeptide or protein, or as a polynucleotide that encodes the aminoacyl-tRNA synthetase.
  • the aminoacyl-tRNA synthetase is derived from an E.
  • the aminoacyl-tRNA synthetase preferentially aminoacylates an E. coli leucyl tRNA (or a variant thereof) with a leucine analog over the naturally-occurring leucine amino acid.
  • the aminoacyl-tRNA synthetase may comprise SEQ ID NO: 1, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.
  • the aminoacyl-tRNA synthetase comprises SEQ ID NO: 1, or a functional fragment or variant thereof, and with one, two, three, four, five or more of the following mutations: (i) a substitution of a glutamine residue at a position corresponding to position 2 of SEQ ID NO: 1, e.g., a substitution by glutamic acid (Q2E); (ii) a substitution of a glutamic acid residue at a position corresponding to position 20 of SEQ ID NO: 1, e.g., a substitution by lysine (E20K), methionine (E20M), or valine (E20V); (iii) a substitution of a methionine residue at a position corresponding to position 40 of SEQ ID NO: 1, e.g., a substitution by isoleucine (M40I) or valine (M40V); (iv) a substitution of a leucine residue at a position corresponding to position 41 of SEQ ID NO:
  • the aminoacyl-tRNA synthetase comprises (i) at least one substitution (e.g., a substitution with a hydrophobic amino acid) at a position corresponding to His537 of SEQ ID NO: 1, (ii) at least one amino acid substitution selected from E20V, E20M, L41V, L41A, Y499H, Y499A, Y527I, Y527V, Y527G, and any combination thereof, (iii) at least one amino acid substitution selected from E20K and L41S and any combination thereof and at least one amino acid substitution selected from M40I, T252A, Y499I, and Y527A, and any combination thereof, or (iv) a combination of two or more of (i), (ii) and (iii), for example, (i) and (ii), (i) and (iii), (ii) and (iii) and (i), (ii) and (i), (ii) and (i
  • the aminoacyl-tRNA synthetase comprises a substitution of a glutamic acid residue at a position corresponding to position 20 of SEQ ID NO: 1, e.g., a substitution with an amino acid other than a Glu or Lys, e.g., a substitution with a hydrophobic amino acid (e.g., Leu, Val, or Met).
  • the aminoacyl- tRNA synthetase comprises a substitution of a leucine residue at a position corresponding to position 41 of SEQ ID NO: 1, e.g., a substitution with an amino acid other than a Leu or Ser, e.g., a substitution with a hydrophobic amino acid other than Leu (e.g., Gly, Ala, Val, or Met).
  • the aminoacyl-tRNA synthetase comprises a substitution of a tyrosine residue at a position corresponding to position 499 of SEQ ID NO: 1, e.g., a substitution with a small hydrophobic amino acid (e.g., Gly, Ala, or Val) or a substitution with a positively charged amino acid (e.g., Lys, Arg, or His).
  • a substitution with a small hydrophobic amino acid e.g., Gly, Ala, or Val
  • a substitution with a positively charged amino acid e.g., Lys, Arg, or His
  • the aminoacyl-tRNA synthetase comprises a substitution of a tyrosine residue at a position corresponding to position 527 of SEQ ID NO: 1, e.g., a substitution with a hydrophobic amino acid other than Ala or Leu (e.g., Gly, Ile, Met, or Val).
  • the tRNA synthetase mutein comprises L41V.
  • the aminoacyl-tRNA synthetase comprises a combination of mutations selected from: (i) Q2E, E20K, M40I, L41S, T252A, Y499I, Y527A, and H537G; (ii) Q2E, E20K, M40V, L41S, T252R, Y499S, Y527L, and H537G; (iii) Q2E, M40I, T252A, Y499I, Y527A, and H537G; (iv) Q2E, E20M, M40I, L41S, T252A, Y499I, Y527A, and H537G; (v) Q2E, E20V, M40I, L41S, T252A, Y499I, Y527A, and H537G; (vi) Q2E, E20K, M40I, L41S, T252A,
  • the aminoacyl-tRNA synthetase comprises the amino acid sequence of any one of SEQ ID NOs: 2-13, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 2- 13.
  • the tRNA synthetase mutein comprises the amino acid sequence of SEQ ID NO: 14, wherein X 2 is Q or E, X 20 is E, K, V or M, X 40 is M, I, or V, X 41 is L, S, V, or A, X 252 is T, A, or R, X 499 is Y, A, I, H, or S, X 527 is Y, A, I, L, or V, and X 537 is H or G, and the tRNA synthetase mutein comprises at least one mutation (for example, 2, 3, 4, 5, 6, 7, 8, 9, or more mutations) relative to SEQ ID NO: 1.
  • the tRNA synthetase mutein comprises the amino acid sequence of SEQ ID NO: 15, wherein X 20 is K, V or M, X 41 is S, V, or A, X 499 is A, I, or H, and X 527 is A, I, or V, and the tRNA synthetase mutein comprises at least one mutation relative to SEQ ID NO: 1.
  • the aminoacyl-tRNA synthetase is derived from an E. coli tryptophanyl-tRNA synthetase and, for example, the aminoacyl-tRNA synthetase preferentially aminoacylates an E.
  • the aminoacyl-tRNA synthetase may comprise SEQ ID NO: 44, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 44.
  • the aminoacyl-tRNA synthetase comprises SEQ ID NO: 44, or a functional fragment or variant thereof, but with one or more of the following mutations: (i) a substitution of a serine residue at a position corresponding to position 8 of SEQ ID NO: 44, e.g., a substitution by alanine (S8A); (ii) a substitution of a valine residue at a position corresponding to position 144 of SEQ ID NO: 44, e.g., a substitution by serine (V144S), glycine (V144G) or alanine (V144A); (iii) a substitution of a valine residue at a position corresponding to position 146 of SEQ ID NO: 44, e.g., a substitution by alanine (V146A), isoleucine (V146I), or cysteine (V146C).
  • the aminoacyl-tRNA synthetase comprises a combination of mutations selected from: (i) S8A, V144S, and V146A, (ii) S8A, V144G, and V146I, (iii) S8A, V144A, and V146A, and (iv) S8A, V144G, and V146C.
  • the aminoacyl-tRNA synthetase comprises the amino acid sequence of any one of SEQ ID NOs: 45-48, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 45- 48.
  • the aminoacyl-tRNA synthetase is derived from an E. coli tyrosyl-tRNA synthetase and, for example, the aminoacyl-tRNA synthetase preferentially aminoacylates an E. coli tyrosyl tRNA (or a variant thereof) with a tyrosine analog over the naturally-occurring tryptophan amino acid.
  • the aminoacyl- tRNA synthetase may comprise SEQ ID NO: 70, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 70, or a functional fragment or variant thereof.
  • the aminoacyl-tRNA synthetase is derived from an M. barkeri pyrrolysyl-tRNA synthetase and, for example, the aminoacyl-tRNA synthetase preferentially aminoacylates an M. barkeri pyrrolysyl tRNA (or a variant thereof) with a pyrrolysine analog over the naturally-occurring pyrrolysine amino acid.
  • the aminoacyl-tRNA synthetase may comprise SEQ ID NO: 101, or an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 101, or a functional fragment or variant thereof.
  • Methods for producing proteins e.g., aminoacyl-tRNA synthetases, are known in the art.
  • DNA molecules encoding a protein of interest can be synthesized chemically or by recombinant DNA methodologies.
  • the resulting DNA molecules encoding the protein interest can be ligated to other appropriate nucleotide sequences, including, for example, expression control sequences, to produce conventional gene expression constructs (i.e., expression vectors) encoding the desired protein. Production of defined gene constructs is within routine skill in the art.
  • Nucleic acids encoding desired proteins e.g, aminoacyl-tRNA synthetases
  • expression vectors which can be introduced into host cells through conventional transfection or transformation techniques.
  • Exemplary host cells are E.
  • Transformed host cells can be grown under conditions that permit the host cells to express the desired protein.
  • Specific expression and purification conditions will vary depending upon the expression system employed. For example, if a gene is to be expressed in E. coli, it is first cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter, e.g., Trp or Tac, and a prokaryotic signal sequence.
  • the expressed protein may be secreted.
  • the expressed protein may accumulate in refractile or inclusion bodies, which can be harvested after disruption of the cells by French press or sonication.
  • the refractile bodies then are solubilized, and the protein may be refolded and/or cleaved by methods known in the art.
  • the engineered gene is to be expressed in eukaryotic host cells, e.g., CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, a poly A sequence, and a stop codon.
  • the vector or gene construct may contain enhancers and introns.
  • the gene construct can be introduced into eukaryotic host cells using conventional techniques.
  • a protein of interest e.g, an aminoacyl-tRNA synthetase
  • an aminoacyl-tRNA synthetase can be produced by growing (culturing) a host cell transfected with an expression vector encoding such a protein under conditions that permit expression of the protein. Following expression, the protein can be harvested and purified or isolated using techniques known in the art, e.g., affinity tags such as glutathione-S-transferase (GST) or histidine tags.
  • affinity tags such as glutathione-S-transferase (GST) or histidine tags.
  • the invention also encompasses nucleic acids encoding aminoacyl-tRNA synthetases disclosed herein.
  • nucleotide sequences encoding leucyl-tRNA synthetase muteins disclosed herein are depicted in SEQ ID NOs: 55-67.
  • the invention provides a nucleic acid comprising the nucleotide sequence of any one of SEQ ID NOs: 55-67, or a nucleotide sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 55-67.
  • the invention also provides a nucleic acid comprising a nucleotide sequence encoding the amino acid sequence encoded by any one of SEQ ID NOs: 55-67, or a nucleotide sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence encoding the amino acid sequence encoded by any one of SEQ ID NOs: 55-67.
  • a nucleotide sequence encoding a tryptophanyl-tRNA synthetase disclosed herein is depicted in SEQ ID NO: 103.
  • the invention provides a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 103, or a nucleotide sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 103.
  • the invention also provides a nucleic acid comprising a nucleotide sequence encoding the amino acid sequence encoded by SEQ ID NO: 103, or a nucleotide sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence encoding the amino acid sequence encoded by SEQ ID NO: 103.
  • a nucleotide sequence encoding a tyrosyl-tRNA synthetase disclosed herein is depicted in SEQ ID NO: 71. Accordingly, the invention provides a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 71, or a nucleotide sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 71.
  • the invention also provides a nucleic acid comprising a nucleotide sequence encoding the amino acid sequence encoded by SEQ ID NO: 71, or a nucleotide sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence encoding the amino acid sequence encoded by SEQ ID NO: 71.
  • a nucleotide sequence encoding a pyrrolysyl-tRNA synthetase disclosed herein is depicted in SEQ ID NO: 102.
  • the invention provides a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 102, or a nucleotide sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 102.
  • the invention also provides a nucleic acid comprising a nucleotide sequence encoding the amino acid sequence encoded by SEQ ID NO: 102, or a nucleotide sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence encoding the amino acid sequence encoded by SEQ ID NO: 102.
  • tRNAs transfer RNAs
  • proteins e.g., antibodies
  • tRNAs transfer RNAs
  • a tRNA molecule delivers an amino acid to a ribosome for incorporation into a growing protein (polypeptide) chain.
  • tRNAs typically are about 70 to 100 nucleotides in length.
  • Active tRNAs contain a 3' CCA sequence that may be transcribed into the tRNA during its synthesis or may be added later during post- transcriptional processing.
  • aminoacyl-tRNA an aminoacyl-tRNA
  • an amino acid can spontaneously migrate from the 2'-hydroxyl group to the 3'-hydroxyl group and vice versa, but it is incorporated into a growing protein chain at the ribosome from the 3'-OH position.
  • a loop at the other end of the folded aa-tRNA molecule contains a sequence of three bases known as the anticodon.
  • this anticodon sequence hybridizes or base-pairs with a complementary three-base codon sequence in a ribosome-bound mRNA
  • the aa-tRNA binds to the ribosome and its amino acid is incorporated into the polypeptide chain being synthesized by the ribosome.
  • all tRNAs that base-pair with a specific codon are aminoacylated with a single specific amino acid
  • the translation of the genetic code is affected by tRNAs.
  • Each of the 61 non-termination codons in an mRNA directs the binding of its cognate aa-tRNA and the addition of a single specific amino acid to the growing polypeptide chain being synthesized by the ribosome.
  • Suppressor tRNAs are modified tRNAs that alter the reading of a mRNA in a given translation system.
  • a suppressor tRNA may read through a codon such as a stop codon, a four base codon, or a rare codon.
  • the use of the word in suppressor is based on the fact, that under certain circumstance, the modified tRNA "suppresses" the typical phenotypic effect of the codon in the mRNA.
  • Suppressor tRNAs typically contain a mutation (modification) in either the anticodon, changing codon specificity, or at some position that alters the aminoacylation identity of the tRNA.
  • the term “suppression activity” refers to the ability of a tRNA, e.g., a suppressor tRNA, to read through a codon (e.g., a premature stop codon) that would not be read through by the endogenous translation machinery in a system of interest.
  • a tRNA e.g., a suppressor tRNA
  • a tRNA comprises an anticodon that hybridizes to a codon selected from UAG (i.e., an “amber” termination codon), UGA (i.e., an “opal” termination codon), and UAA (i.e., an “ochre” termination codon).
  • a tRNA comprises an anticodon that hybridizes to a non-standard codon, e.g., a 4- or 5-nucleotide codon. Examples of four base codons include AGGA, CUAG, UAGA, and CCCU. Examples of five base codons include AGGAC, CCCCU, CCCUC, CUAGA, CUACU, and UAGGC.
  • tRNAs comprising an anticodon that hybridizes to a non-standard codon, e.g., a 4- or 5-nucleotide codon
  • a non-standard codon e.g., a 4- or 5-nucleotide codon
  • methods of using such tRNAs to incorporate unnatural amino acids into proteins are described, for example, in Moore et al. (2000) J. MOL. BIOL.298:195; Hohsaka et al. (1999) J. AM. CHEM. SOC. 121:12194; Anderson et al. (2002) CHEMISTRY AND BIOLOGY 9:237-244; Magliery (2001) J. MOL. BIOL.307: 755-769; and PCT Publication No. WO2005/007870.
  • tRNA includes variants having one or more mutations (e.g., nucleotide substitutions, deletions, or insertions) relative to a reference (e.g., a wild-type) tRNA sequence.
  • a tRNA may comprise, consist, or consist essentially of, a single mutation (e.g., a mutation contemplated herein), or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more than 15 mutations (e.g., mutations contemplated herein).
  • a tRNA may comprise, consist, or consist essentially 1-15, 1-10, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-15, 2-10, 2-7, 2-6, 2-5, 2-4, 2-3, 3-15, 3-10, 3-7, 3-6, 3-5, or 3-4 mutations (e.g., mutations contemplated herein).
  • a variant suppressor tRNA has increased activity to incorporate an unnatural amino acid (e.g., an unnatural amino acid contemplated herein) into a mammalian protein relative to a counterpart wild-type suppressor tRNA (in this context, a wild-type suppressor tRNA refers to a suppressor tRNA that corresponds to a wild-type tRNA molecule but for any modifications to the anti-codon region to impart suppression activity).
  • an unnatural amino acid e.g., an unnatural amino acid contemplated herein
  • a wild-type suppressor tRNA refers to a suppressor tRNA that corresponds to a wild-type tRNA molecule but for any modifications to the anti-codon region to impart suppression activity.
  • the activity of the variant suppressor tRNA may be increased relative to the wild type suppressor tRNA, for example, by about 2.5 to about 200 fold, about 2.5 to about 150 fold, about 2.5 to about 100 fold about 2.5 to about 80 fold, about 2.5 to about 60 fold, about 2.5 to about 40 fold, about 2.5 to about 20 fold, about 2.5 to about 10 fold, about 2.5 to about 5 fold, about 5 to about 200 fold, about 5 to about 150 fold, about 5 to about 100 fold, about 5 to about 80 fold, about 5 to about 60 fold, about 5 to about 40 fold, about 5 to about 20 fold, about 5 to about 10 fold, about 10 to about 200 fold, about 10 to about 150 fold, about 10 to about 100 fold, about 10 to about 80 fold, about 10 to about 60 fold, about 10 to about 40 fold, about 10 to about 20 fold, about 20 to about 200 fold, about 20 to about 150 fold, about 20 to about 100 fold, about 20 to about 80 fold, about 20 to about 60 fold, about 20 to about 40 fold, about 40 to about 200 fold, about 40 to about 150 fold, about 40 to
  • the tRNA may function in vitro or in vivo and can be provided to a translation system (e.g., an in vitro translation system or a cell) as a mature tRNA (e.g., an aminoacylated tRNA), or as a polynucleotide that encodes the tRNA.
  • a tRNA may be derived from a bacterial source, e.g., Escherichia coli, Thermus thermophilus, or Bacillus stearothermphilus.
  • a tRNA may also be derived from an archaeal source, e.g, from the Methanosarcinacaea or Desulfitobacterium families, any of the M.
  • eukaryotic sources can also be used, for example, plants, algae, protists, fungi, yeasts, or animals (e.g., mammals, insects, arthropods, etc.).
  • the tRNA is derived from an E.
  • coli leucyl tRNA and, for example, is preferentially charged with a leucine analog over the naturally-occurring leucine amino acid by an aminoacyl-tRNA synthetase derived from an E. coli leucyl-tRNA synthetase, e.g., an aminoacyl-tRNA synthetase contemplated herein.
  • the tRNA may comprise, consist essentially of, or consist of the nucleotide sequence of any one of SEQ ID NOs: 16-43, or a nucleotide sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 16-43.
  • the tRNA is derived from an E. coli tryptophanyl tRNA and, for example, is preferentially charged with a tryptophan analog over the naturally- occurring tryptophan amino acid by an aminoacyl-tRNA synthetase derived from an E.
  • the tRNA may comprise, consist essentially of, or consist of the nucleotide sequence of any one of SEQ ID NOs: 49-54 or 108-113, or a nucleotide sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 49-54 or 108-113.
  • the tRNA is derived from an E.
  • coli tyrosyl tRNA and, for example, is preferentially charged with a tyrosine analog over the naturally- occurring tyrosine amino acid by an aminoacyl-tRNA synthetase derived from an E. coli tyrosyl-tRNA synthetase, e.g., an aminoacyl-tRNA synthetase contemplated herein.
  • the tRNA may comprise, consist essentially of, or consist of the nucleotide sequence of any one of SEQ ID NOs: 68-69 or 104-105, or a nucleotide sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 68-69 or 104-105.
  • the tRNA is derived from a M.
  • barkeri pyrrolysyl tRNA is preferentially charged with a pyrrolysine analog over the naturally-occurring pyrrolysine amino acid by an aminoacyl-tRNA synthetase derived from a M. barkeri pyrrolysyl-tRNA synthetase, e.g., an aminoacyl-tRNA synthetase contemplated herein.
  • the tRNA may comprise, consist essentially of, or consist of the nucleotide sequence of any one of SEQ ID NOs: 72-100 or 106-107, or a nucleotide sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 72-100 or 106-107.
  • a tRNA comprises, consists essentially of, or consists of a nucleotide sequence including one or more thymines (T)
  • a tRNA is also contemplated that comprises, consists essentially of, or consists of the same nucleotide sequence including a uracil (U) in place of one or more of the thymines (T), or a uracil (U) in place of all the thymines (T).
  • a tRNA comprises, consists essentially of, or consists of a nucleotide sequence including one or more uracils (U)
  • a tRNA is also contemplated that comprises, consists essentially of, or consists of a nucleotide sequence including a thymine (T) in place of the one or more of the uracils (U), or a thymine (T) in place of all the uracils (U).
  • T thymine
  • additional modifications to the bases can be present.
  • a tRNA may be aminoacylated (i.e., charged) with a desired unnatural amino acid (UAA) by any method, including enzymatic or chemical methods.
  • Enzymatic molecules capable of charging a tRNA include aminoacyl-tRNA synthetases, e.g., aminoacyl-tRNA synthetases disclosed herein. Additional enzymatic molecules capable of charging tRNA include ribozymes, for example, as described in Illangakekare et al.
  • Chemical aminoacylation methods include those described in Hecht (1992) ACC. CHEM. RES.25:545, Heckler et al. (1988) BIOCHEM.1988, 27:7254, Hecht et al. (1978) J. BIOL. CHEM.253:4517, Cornish et al. (1995) ANGEW. CHEM. INT. ED.
  • tRNAs, aminoacyl-tRNA synthetases, or any other molecules of interest may be expressed in a cell of interest by incorporating a gene encoding the molecule into an appropriate expression vector.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • tRNAs, aminoacyl-tRNA synthetases, or any other molecules of interest may be introduced to a cell of interest by incorporating a gene encoding the molecule into an appropriate transfer vector.
  • transfer vector refers to a vector comprising a recombinant polynucleotide which can be used to deliver the polynucleotide to the interior of a cell. It is understood that a vector may be both an expression vector and a transfer vector.
  • Vectors e.g., expression vectors or transfer vectors
  • Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular target nucleic acid.
  • the vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both (including but not limited to, shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems.
  • the vector comprises a regulatory sequence or promoter operably linked to the nucleotide sequence encoding the suppressor tRNA and/or the tRNA synthetase.
  • operably linked refers to a linkage of polynucleotide elements in a functional relationship.
  • a nucleic acid sequence is "operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a gene if it affects the transcription of the gene.
  • Operably linked nucleotide sequences are typically contiguous.
  • enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths
  • some polynucleotide elements may be operably linked but not directly flanked and may even function in trans from a different allele or chromosome.
  • Exemplary promoters which may be employed include, but are not limited to, the retroviral LTR, the SV40 promoter, the human cytomegalovirus (CMV) promoter, the U6 promoter, the EF1 ⁇ promoter, the CAG promoter, the H1 promoter, the UbiC promoter, the PGK promoter, the 7SK promoter, a pol II promoter, a pol III promoter, or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and ⁇ -actin promoters).
  • CMV human cytomegalovirus
  • a vector comprises a nucleotide sequence encoding an aminoacyl-tRNA synthetase operably linked to a CMV or an EF1 ⁇ promoter and/or a nucleotide sequence encoding a suppressor tRNA operably linked to a U6 or an H1 promoter.
  • the vector is a viral vector.
  • virus is used herein to refer to an obligate intracellular parasite having no protein-synthesizing or energy- generating mechanism.
  • exemplary viral vectors include retroviral vectors (e.g., lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpesviruses vectors, epstein- barr virus (EBV) vectors, polyomavirus vectors (e.g., simian vacuolating virus 40 (SV40) vectors), poxvirus vectors, and pseudotype virus vectors.
  • retroviral vectors e.g., lentiviral vectors
  • adenoviral vectors e.g., adenoviral vectors, adeno-associated viral vectors, herpesviruses vectors, epstein- barr virus (EBV) vectors, polyomavirus vectors (e.g., simian vacuolating virus 40 (SV40) vectors), poxvirus
  • the viral vector is a DNA virus vector.
  • DNA viruses include parvoviruses (e.g., adeno-associated viruses), adenoviruses, asfarviruses, herpesviruses (e.g., herpes simplex virus 1 and 2 (HSV-1 and HSV-2), epstein-barr virus (EBV), cytomegalovirus (CMV)), papillomoviruses (e.g., HPV), polyomaviruses (e.g., simian vacuolating virus 40 (SV40)), and poxviruses (e.g., vaccinia virus, cowpox virus, smallpox virus, fowlpox virus, sheeppox virus, myxoma virus).
  • parvoviruses e.g., adeno-associated viruses
  • adenoviruses e.g., asfarviruses
  • herpesviruses e.g., her
  • the viral vector is a RNA virus vector.
  • RNA viruses include bunyaviruses (e.g., hantavirus), coronaviruses, flaviviruses (e.g., yellow fever virus, west nile virus, dengue virus), hepatitis viruses (e.g., hepatitis A virus, hepatitis C virus, hepatitis E virus), influenza viruses (e.g., influenza virus type A, influenza virus type B, influenza virus type C), measles virus, mumps virus, noroviruses (e.g., Norwalk virus), poliovirus, respiratory syncytial virus (RSV), retroviruses (e.g., human immunodeficiency virus-1 (HIV-1)) and toroviruses.
  • bunyaviruses e.g., hantavirus
  • coronaviruses e.g., flaviviruses (e.g., yellow fever virus, west nile virus,
  • host cells or cell lines e.g., prokaryotic or eukaryotic host cells or cell lines
  • the nucleic acid encoding the engineered tRNA and aminoacyl-tRNA synthetase can be expressed in an expression host cell either as an autonomously replicating vector within the expression host cell (e.g., a plasmid, or viral particle) or via a stable integrated element or series of stable integrated elements in the genome of the expression host cell, e.g., a mammalian host cell.
  • Host cells are genetically engineered (including but not limited to, transformed, transduced or transfected), for example, using nucleic acids or vectors disclosed herein.
  • one or more vectors include coding regions for an orthogonal tRNA, an orthogonal aminoacyl-tRNA synthetase, and, optionally, a protein (e.g., an antibody) to be modified by the inclusion of one or more UAAs, which are operably linked to gene expression control elements that are functional in the desired host cell or cell line.
  • the genes encoding tRNA synthetase and tRNA and an optional selectable marker can be integrated in a transfer vector (e.g., a plasmid, which can be linearized prior to transfection), where for example, the genes encoding the tRNA synthetase can be under the control of a polymerase II promoter (e.g., CMV, EF1 ⁇ , UbiC, or PGK, e.g., CMV or EF1 ⁇ ) and the genes encoding the tRNA can be under the control of a polymerase III promoter (e.g., U6, 7SK, or H1, e.g., U6).
  • a polymerase II promoter e.g., CMV, EF1 ⁇ , UbiC, or PGK, e.g., CMV or EF1 ⁇
  • a polymerase III promoter e.g., U6, 7SK, or H1, e.g., U6
  • the vectors are transfected into cells and/or microorganisms by standard methods including electroporation or infection by viral vectors, and clones can be selected via expression of the selectable marker (for example, by antibiotic resistance).
  • exemplary prokaryotic host cells or cell lines include cells derived from a bacteria, e.g., Escherichia coli, Thermus thermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, and Pseudomonas putida.
  • Exemplary eukaryotic host cells or cell lines include cells derived from a plant (e.g., a complex plant such as a monocot or dicot), an algae, a protist, a fungus, a yeast (including Saccharomyces cerevisiae), or an animal (including a mammal, an insect, an arthropod, etc.).
  • a plant e.g., a complex plant such as a monocot or dicot
  • an algae e.g., a complex plant such as a monocot or dicot
  • a protist e.g., a fungus
  • yeast including Saccharomyces cerevisiae
  • animal including a mammal, an insect, an arthropod, etc.
  • Additional exemplary host cells or cell lines include HEK293, HEK293T, Expi293, CHO, CHOK1, Sf9, Sf21, HeLa, U20S, A549, HT1080, CAD, P19, NIH 3T3, L929, N2a, MCF-7, Y79, SO- RB50, HepG2, DUKX-X11, J558L, BHK, COS, Vero, NS0, or ESCs. It is understood that a host cell or cell line can include individual colonies, isolated populations (monoclonal), or a heterogeneous mixture of cells.
  • a contemplated cell or cell line includes, for example, one or multiple copies of an orthogonal tRNA/aminoacyl-tRNA synthetase pair, optionally stably maintained in the cell’s genome or another piece of DNA maintained by the cell.
  • the cell or cell line may contain one or more copies of (i) a tryptophanyl tRNA/aminoacyl-tRNA synthetase pair (wild type or engineered) stably maintained by the cell, and/or (ii) a leucyl tRNA/aminoacyl-tRNA synthetase pair (wild-type or engineered) stably maintained by the cell.
  • the cell line is a stable cell line and the cell line comprises a genome having stably integrated therein (i) a nucleic acid sequence encoding an aminoacyl-tRNA synthetase (e.g., a prokaryotic tryptophanyl-tRNA synthetase mutein capable of charging a tRNA with an unnatural amino acid or a prokaryotic leucyl- tRNA synthetase mutein capable of charging a tRNA with an unnatural amino acid, e.g., a tRNA synthetase mutein disclosed herein); and/or (ii) a nucleic acid sequence encoding a suppressor tRNA (e.g., prokaryotic suppressor tryptophanyl-tRNA capable of being charged with an unnatural amino acid or prokaryotic suppressor leucyl-tRNA capable of being charged with an unnatural amino acid, e.
  • nucleic acid encoding a tRNA and/or an aminoacyl- tRNA synthetase into the genome of a cell of interest, or to stably maintain the nucleic acid in DNA replicated by the cell that is outside of the genome, are well known in the art.
  • the nucleic acid encoding the tRNA and/or an aminoacyl-tRNA synthetase can be provided to the cell in an expression vector, transfer vector, or DNA cassette, e.g., an expression vector, transfer vector, or DNA cassette disclosed herein.
  • the expression vector transfer vector, or DNA cassette encoding the tRNA and/or aminoacyl-tRNA synthetase can contain one or more copies of the tRNA and/or aminoacyl-tRNA synthetase optionally under the control of an inducible or constitutively active promoter.
  • the expression vector, transfer vector, or DNA cassette may, for example, contain other standard components (enhancers, terminators, etc.).
  • nucleic acid encoding the tRNA and the nucleic acid encoding the aminoacyl-tRNA synthetase may be on the same or different vector, may be present in the same or different ratios, and may be introduced into the cell, or stably integrated in the cellular genome, at the same time or sequentially.
  • One or multiple copies of a DNA cassette encoding the tRNA and/or aminoacyl-tRNA synthetase can be integrated into a host cell genome or stably maintained in the cell using a transposon system (e.g., PiggyBac), a viral vector (e.g., a lentiviral vector or other retroviral vector), CRISPR/Cas9 based recombination, electroporation and natural recombination, a BxB1 recombinase system, or using a replicating/maintained piece of DNA (such as one derived from Epstein-Barr virus).
  • a transposon system e.g., PiggyBac
  • a viral vector e.g., a lentiviral vector or other retroviral vector
  • CRISPR/Cas9 based recombination e.g., a lentiviral vector or other retroviral vector
  • electroporation and natural recombination
  • a selectable marker can be used.
  • exemplary selectable markers include zeocin, puromycin, neomycin, dihydrofolate reductase (DHFR), glutamine synthetase (GS), mCherry-EGFP fusion, or other fluorescent proteins.
  • a gene encoding a selectable marker protein may include a premature stop codon, such that the protein will only be expressed if the cell line is capable of incorporating a UAA at the site of the premature stop codon.
  • a host cell or cell line including two or more tRNA/aminoacyl-tRNA synthetase pairs one can use multiple identical or distinct UAA directing codons in order to identify host cells or cell lines which have incorporated multiple copies of the two or more tRNA/aminoacyl-tRNA synthetase pairs through iterative rounds of genomic integration and selection.
  • Host cells or cell lines which contain enhanced UAA incorporation efficiency, low background, and decreased toxicity can first be isolated via a selectable marker containing one or more stop codons.
  • the host cells or cell lines can be subjected to a selection scheme to identify host cells or cell lines which contain the desired copies of tRNA/aminoacyl-tRNA synthetase pairs and express a gene of interest (either genomically integrated or not) containing one or more stop codons. Protein expression may be assayed using any method known in the art, including for example, Western blot using an antibody that binds the protein of interest or a C-terminal tag.
  • the host cells or cell lines be cultured in conventional nutrient media modified as appropriate for such activities as, for example, screening steps, activating promoters or selecting transformants. These cells can optionally be cultured into transgenic organisms.
  • Other useful references e.g.
  • compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.
  • FIGURE 7 As outlined in FIGURE 7, four different antibodies (Trastuzumab tested twice (Trastuzumab A and Trastuzumab B), a non-Trastuzumab IgG1, a non-Trastuzumab bispecific antibody, and a second non-Trastuzumab IgG1) were mutated to contain either a TAG or TGA stop codon at one or multiple sites in order to introduce unnatural amino acids as previously described, using transient transfection screening methods. Sites of consideration for the present studies are shown in FIGURE 2. These antibodies were subsequently conjugated with an exemplary partner payload, identified from the list provided in FIGURE 7.
  • Antibodies and antibody drug conjugates were characterized with the parameters and assays outlined in FIGURE 7.
  • Antibodies containing the unnatural amino acid LCA [00270] The following protocol was used for expression of antibodies containing LCA, and conjugation of molecules to the antibodies with a target drug-antibody ratio of 2 to 4 (DAR2-4). [00271] All monoclonal antibody expression was performed using the Expi293 Expression System according to the manufacturer’s instructions. Briefly, before transfection, cells were split to a density of 2.7 ⁇ 10 6 to 3.0 ⁇ 10 6 cells/ml. An unnatural amino acid (UAA), LCA, was added to achieve a final concentration of 0.25 to 0.5 mM.
  • UAA unnatural amino acid
  • plasmid mix (equal parts suppressor plasmid, heavy chain plasmid, and light chain plasmid) was used for transfection into 1 L cell culture.
  • Suppressor plasmid contained anywhere from 1 to 20 copies of leucyl tRNA (Leu-tRNA.h1; SEQ ID NO: 19) or leucyl synthetase (LeuRS.v1; SEQ ID NO: 2).
  • Heavy chain or light chain plasmids were mutated to contain a TAG (LCA) stop codon for the incorporation of the UAA. Plasmids were diluted in 50 ml Opti-MEM medium.
  • a PEI stock solution was incubated at room temperature for 3 minutes, subsequently diluted to achieve a 6:1 PEI:DNA final ratio, and incubated for 15 minutes at room temperature.
  • the Plasmid:PEI complex was then added dropwise to the culture.
  • 0.25 to 1 mM LCA was added to the cells.
  • Cells were incubated on an orbital shaking platform at 37 °C with 8% CO 2 at a speed of 80 to 125 rpm for 5 to 8 days.
  • Protein was purified using a PrismA column (Cytiva: 17549801) for use in characterization assays described herein.
  • HTP-containing antibodies containing unnatural amino acid HTP
  • the suppressor plasmid used contained the tryptophanyl tRNA (Trp-tRNA-UCA; SEQ ID NO: 51) / aaRS (trpRS.h14; SEQ ID NO: 45) pair rather than leucyl, as well as a TGA stop codon rather than a TAG stop codon, in order to direct the incorporation of the UAA into the antibody site of interest.
  • HTP was added to the cells for a final concentration of 0.25 to 1 mM. Yields of HTP-containing proteins are outlined in FIGURE 31.
  • Site-specific cysteine mutants were prepared according to techniques known in the art, to allow for direct comparison to present UAA sites of interest, such as T198.
  • the heavy chain of trastuzumab was mutated to contain a T198-cysteine mutation, and was transfected and expressed in Expi293 cells, without suppressor plasmid or UAA addition, as described above.
  • cysteine sites were conjugated by substitution of cysteine at a site of interest using molecular biology techniques known in the art.
  • the mAb was buffer exchanged to 50 mM HEPES, pH 7.2 with 2 mM EDTA.
  • TCEP tris(2- carboxyethyl)phosphine
  • LCA-containing antibodies disclosed herein to introduce payloads such as PEG4/PEG8 linkers, valine-citrulline (VC) linkers, small hydrophobic payloads, peptide-based payloads, nucleic acids (such as locked nucleic acids) and other hydrophobic payloads.
  • payloads such as PEG4/PEG8 linkers, valine-citrulline (VC) linkers, small hydrophobic payloads, peptide-based payloads, nucleic acids (such as locked nucleic acids) and other hydrophobic payloads.
  • Click Chemistry conjugation protocol and HIC characterization [00280] LCA-containing antibodies were diluted to a final concentration of 1 mg/ml to 5 mg/ml in PBS.10 mM to 50 mM of payload stock solutions were made in buffer or organic solvent (DMSO, DMF, etc.) as appropriate.
  • FIGURE 14 and FIGURE 15 depict the average DAR post conjugation of LCA- containing antibodies with a variety of payloads (exemplary payloads are shown in FIGURE 8, FIGURE 9, FIGURE 10, and FIGURE 11).
  • HIC metrics As shown in FIGURE 14 and FIGURE 15, all samples were analyzed with a MabPac Butyl column.
  • a mobile phase A was 1.5 M ammonium sulfate, 25 mM sodium phosphate dibasic, pH 7 and a mobile phase B was 25 mM sodium phosphate, pH 7.
  • the samples were eluted with a flow rate of 0.8 mL/min and at a column temperature of 25 o C.
  • the average DAR and distribution profile were determined by peak area percentage of each species.
  • the theoretical DAR is 2.0, with an acceptable DAR (per industry standards) being as close to 2.0 as possible.
  • the industry standard DAR is greater than 1.5, and, importantly, the majority of the sites identified in the present study achieved this threshold for any payload, demonstrating the versatility of these sites to attach any biologically or chemically acceptable payload.
  • multiple LCAs were introduced into the antibody scaffold at sites specified in FIGURE 32A and FIGURE 32B.
  • FIGURE 4 depicts raw data for a number of the Monomethyl auristatin D (MMAD)-payload-based LCA conjugates.
  • MMAD Monomethyl auristatin D
  • FIGURE 5 depicts mass spectrometry results.
  • FIGURE 31 provides results for both IgG1a antibodies with HTP-conjugates and bispecific antibodies containing HTP- conjugates. In each instance, the target DAR was achieved, demonstrating the versatility of the sites listed in FIGURE 31 for the incorporation and conjugation of HTP-containing UAAs.
  • Multi-drug conjugation For introduction of multiple distinct UAAs into the same protein (e.g., introduction of LCA and HTP, as shown in FIGURE 32A and FIGURE 32B), a conjugation was performed in a one pot reaction.
  • FIGURE 16 and FIGURE 17 depict aggregation, as measured by SEC, for LCA-based conjugates. As shown in FIGURES 16-17, most sites resulted in little to no protein aggregation (e.g., less than or equal to 1% aggregation). Locked-nucleic-acid (LNA)-containing mAbs contained slightly more aggregation, peaking at about 7%; it is believed this aggregation value could be significantly decreased with the introduction of wash and/or additional clean-up steps.
  • LNA Locked-nucleic-acid
  • FIGURE 31 demonstrates aggregation data for HTP-containing antibodies and their conjugates. For these antibodies, aggregation was slightly higher than that observed with LCA (about 5% instead of about 1%), demonstrating the impact of incorporating different UAAs at different sites.
  • HTP at T198 resulted in the least amount of aggregation, 1%, which was similar to the LCA aggregation propensities described above. The majority of these aggregation propensities pass industry thresholds and can be further improved with additional cleanup steps.
  • SEC of multisite conjugates single and multi-drug were analyzed, as shown in FIGURE 32A (Trastuzumab) and FIGURE 32B (non-Trastuzumab IgG1 and bispecific antibody). All combinations of the single site LCA conjugates (first nine) that demonstrated the least aggregation resulted in little to no aggregation when combined for DAR4+ or multiple distinct UAAs.
  • Example 3 Predictive functionality assays [00289] This example describes characterization of the presently described antibodies and antibody conjugates in preclinical assays chosen based on their ability to predict efficacy in in vivo models. These assays included human plasma stability assays, cathepsin B cleavage assays, and in vitro cytotoxicity assays. Human plasma stability assay [00290] Human plasma (Molecular Innovations, Cat.
  • HPLA-SER-GF-100ML was spiked with a final antibody drug conjugate (ADC) concentration of 0.2 mg/mL and subsequently incubated at 37 °C in 100 ⁇ L aliquots for up to 72 hours; aliquots were immediately flash frozen and stored at -80 °C until further analysis.
  • ADC antibody drug conjugate
  • plasma- incubated ADCs were thawed and subjected to affinity capture with Protein G magnetic beads. The eluted samples containing purified incubated ADC were then analyzed by HIC as described above. Stability was measured by calculating the percentage of ADC remaining post-human plasma incubation.
  • LCA incorporation sites such as T198 and A121
  • T198 and A121 have robust stability in plasma with a variety of cleavable and non-cleavable payloads.
  • some sites, such as HC-T158 showed a lack of stability, even with non-cleavable payloads in human plasma, highlighting the importance of analysis across multiple sites of incorporation.
  • Cathepsin B assay [00291] To measure the stability of cleavable linkers based on site of incorporation, activated Cathepsin B was incubated with LCA-conjugated ADCs in reaction buffer (25 mM sodium acetate and 1 mM EDTA at pH 5.0).
  • MMAE Monomethyl auristatin E
  • Non-toxic SM payloads were linked via cleavable linkers, but some sites such as L154 and K213 were found not cleavable in this assay.
  • T109, A121, and T198 represent robust sites that can accommodate a variety of payloads which require cathepsin based cleavage.
  • HER2 Elisa Binding Assay An ELISA binding assay was employed to compare the effect of UAA incorporation and conjugation of anti-HER2 ADCs.5 ⁇ g of human ErbB2 receptor Fc conjugate was captured on a clear 96-well plate for 1 hour at room temperature. After blocking with 3% milk, ADC samples were added at dilutions ranging from 0 to 100 nM and incubated for 1 hour at room temperature. After washing with 0.05% Tween-20 buffer, goat anti-human ⁇ -HRP conjugate was added at 1:1,000 dilution in blocking buffer and incubated for 1 hour at room temperature. QuantaBlu Fluorogenic Peroxidase Substrate was used for the binding quantification.
  • FIGURE 22A and FIGURE 22B the incorporation of UAAs at sites T109, A121, A143, and T198, and conjugation of these resides with DBCO- PEG4/PEG8-MMAD, maintained sub-nanomolar binding affinities. These data demonstrate that at the particular sites tested there is minimal perturbation of the antibody’s antigen- binding ability.
  • Example 4 In vitro cytotoxicity
  • This example describes the in vitro cytotoxicity of anti-HER2-MMAD conjugates, which are used to depict the effect of site of incorporation and linker conjugation on the overall activity of the ADC conjugates of the present disclosure.
  • HER2+ (NCI-N87) and HER2- (NCI-H520) cell lines were selected for testing of ADCs.8-point serial dilutions of each test sample were tested on the selected cell line(s). After 96 hours incubation in the presence of ADCs, cell viability was measured using a CellTiter-Glo® assay according to the manufacturer’s instructions.
  • FIGURE 23 depicts a table of the IC50 for all tested sites containing DBCO-PEG4/PEG8-MMAD conjugates and FIGURE 24A and FIGURE 24B together depict all cytotoxicity traces.
  • FIGURE 25, FIGURE 26, FIGURE 27, and FIGURE 28 depict T198, A143, A121, and T109 curves in comparison to a T198 MC-MMAD cysteine site-specific mutant as described in Example 1 above, in NCI-N87 cells (FIGURE 25A, FIGURE 26A, FIGURE 27A, and FIGURE 28A) and NCI-H520 cells (FIGURE 25B, FIGURE 26B, FIGURE 27B, and FIGURE 28B).
  • NCI-N87 cells FIGURE 25A, FIGURE 26A, FIGURE 27A, and FIGURE 28A
  • NCI-H520 cells FIGGURE 25B, FIGURE 26B, FIGURE 27B, and FIGURE 28B.
  • PEG8 payload outcompeted PEG4 payload, with slight variations observed between sites (FIGURE 29A and FIGURE 29B, and FIGURE 30A and FIGURE 30B).

Abstract

L'invention concerne de manière générale des anticorps modifiés contenant des acides aminés non naturels (UAA) et des procédés de fabrication et d'utilisation de tels anticorps.
EP21867714.4A 2020-09-10 2021-09-10 Anticorps contenant des acides aminés non naturels et procédés de fabrication et d'utilisation de ceux-ci Pending EP4210725A1 (fr)

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EP1724282B1 (fr) * 1997-05-21 2013-05-15 Merck Patent GmbH Procédé de production de protéines non-immunogènes
ES2865473T3 (es) * 2013-07-10 2021-10-15 Sutro Biopharma Inc Anticuerpos que comprenden múltiples residuos de aminoácidos no naturales sitio-específicos, métodos para su preparación y métodos de uso
US10717975B2 (en) * 2016-06-03 2020-07-21 Trustees Of Boston College Universal platform for genetic code expansion

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